Pathogen inactivation assay

ABSTRACT

The present invention relates to methods for determining the level of potentially active biological pathogens, such as viruses, bacteria (including inter- and intracellular bacteria, such as mycoplasmas, ureaplasmas, nanobacteria, chlamydia, rickettsias), fungi (including yeasts) and single cell parasites, which may be found in a biological material. The present invention particularly relates to methods of determining the level of potentially active biological pathogens in a biological material using quantitative PCR.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to methods for determining the level of potentially active biological pathogens, such as viruses, bacteria (including inter- and intracellular bacteria, such as mycoplasmas, ureaplasmas, nanobacteria, chlamydia, rickettsias), fungi (including yeasts), and single- and multi-cell parasites, which may be found in a biological material. The present invention particularly relates to methods of determining the level of potentially active biological pathogens in a biological material using quantitative PCR, and so may be particularly useful for determining the effectiveness of a sterilization process that has been applied to the biological material.

[0003] 2. Background of the Related Art

[0004] Many biological materials that are prepared for human, veterinary, diagnostic and/or experimental use may contain unwanted and potentially dangerous biological pathogens, such as viruses, bacteria, in both vegetative and spore states, (including inter- and intracellular bacteria, such as mycoplasmas, ureaplasmas, nanobacteria, chlamydia, rickettsias), fungi (including yeasts), and single- and multi-cell parasites. This may also be true of biological materials that are produced in or exported from locations where certain biological pathogens may exist to locations where those biological pathogens are not endemic. Consequently, it is of utmost importance that any biological pathogen in the biological material be inactivated before the material is used. This is especially critical when the biological material is to be administered directly to a patient, for example in tissue implants, blood transfusions, blood factor replacement therapy, organ transplants, and other forms of human and/or other animal therapy corrected or treated by surgical implantation, intravenous, intramuscular or other forms of injection or introduction. This is also critical for the various biological materials that are prepared in media or via the culture of cells, or recombinant cells which contain various types of plasma and/or plasma derivatives or other biological materials and which may be subject to mycoplasmal, prion, ureaplasmal, bacterial, viral and/or other biological pathogens.

[0005] All living cells and multi-cellular organisms can be infected with viruses and other pathogens. Thus, the products of unicellular natural or recombinant organisms or tissues virtually always carry a risk of pathogen contamination. In addition to the risk that the producing cells or cell cultures may be infected, the processing of these and other biological materials also creates opportunities for environmental contamination. The risks of infection are more apparent for multi-cellular natural and recombinant organisms, such as transgenic animals.

[0006] Interestingly, even products from species as different from humans as transgenic plants carry risks, both due to processing contamination as described above, and from environmental contamination in the growing facilities, which may be contaminated by pathogens from the environment or infected organisms that co-inhabit the facility along with the desired plants. For example, a crop of transgenic corn grown in a field could be expected to be exposed to rodents such as mice during the growing season. Mice can harbor serious human pathogens such as the frequently fatal Hanta virus. Since these animals would be undetectable in the growing crop, viruses shed by the animals could be carried into the transgenic material at harvest. Indeed, such rodents are notoriously difficult to control, and may gain access to a crop during sowing, growth, harvest or storage. Likewise, contamination from overflying or perching birds has the potential to transmit such serious pathogens as the causative agent for psittacosis. Thus, any biological material, regardless of its source, may harbor serious pathogens that must be removed or inactivated prior to administration of the material to a recipient human or other animal.

[0007] Accordingly, many procedures for producing human compatible biological materials have involved methods that screen or test the biological materials for one or more particular biological pathogens rather than removal or inactivation of the pathogen(s) from the biological material. The typical protocol for disposition of materials that test positive for a biological pathogen simply is non-use/discarding of that material.

[0008] Examples of screening procedures for contaminants include testing for a particular virus in human blood and tissues from donors. Such procedures, however, are not always reliable and are not able to detect the presence of certain viruses, and prions, particularly those present in very low numbers. This reduces the value, certainty, and safety of such tests in view of the consequences associated with a false negative result, which can be life threatening in certain cases, for example in the case of Acquired Immune Deficiency Syndrome (AIDS). Furthermore, in some instances it can take weeks, if not months, to determine whether or not the material is contaminated. Moreover, to date, there is no commercially available, reliable test or assay for identifying ureaplasmas, mycoplasmas, and chlamydia within a biological material that is fully suitable for screening out potential donors or infected material (Advances in Contraception 10(4):309-315(1994)). This serves to heighten the need for an effective means of destroying ureaplasmas, mycoplasmas, chlamydia, etc., within a biological material, while still retaining the desired activity of that material. Therefore, it is highly desirable to apply techniques that kill or inactivate biological pathogens during and/or after manufacturing and/or harvesting the biological material.

[0009] More recent efforts have focussed on methods to remove or inactivate contaminants in products intended for use in humans and other animals. Particularly useful methods are those that alter the genetic material of a biological pathogen, such as the addition of chemical inactivants or sensitizers to a biological material or irradiation of a biological material.

[0010] The use of chemical inactivants or sensitizers involves the addition of noxious agents which bind to the DNA/RNA of the virus, and which are activated either by UV or other radiation. This radiation produces reactive intermediates and/or free radicals which bind to the DNA/RNA of the virus, break the chemical bonds in the backbone of the DNA/RNA, and/or cross-link or complex it in such a way that the virus can no longer replicate.

[0011] Irradiating a biological material with ionizing radiation, such as gamma, UV or e-beam radiation, is another method of sterilizing a product. The direct effects of gamma radiation are particularly useful for destroying the genetic material within viruses and bacteria, particularly when given in total doses of at least 25 kGy (See Keathly, et al., “Is There Life After Irradiation? Part 2,” BioPharm July-August, 1993, and Leitman, “Use of Blood Cell Irradiation in the Prevention of Post Transfusion Graft-vs-Host Disease,” Transfusion Science 10:219-239(1989)).

[0012] The use of such sterilization methods does not, however, remove any biological pathogens from the sterilized biological material. Rather, these methods render inactive any biological pathogens that may be present in the biological material by altering the genetic material within the pathogen, including cleaving, deleting, oxidizing, reducing, covalently bonding, cross-linking and/or complexing that genetic material or a component thereof.

[0013] For many potentially active biological pathogens, a single modification in their genome may be sufficient to render them inactive. Significantly, most presently available tests for the detection or quantification of biological pathogens, such as ELISA tests for surface antigens, will not indicate that the biological pathogen has been rendered inactive. Moreover, conventional genetic detection tests, such as the PCR reaction described below, examine only a small portion of the genome and may fail to detect sites of alteration that render the biological pathogen inactive, irrespsective of whether there ate several such sites or only one. In practice, these tests may frequently report a high level of false positive results, leading to inappropriate product quarantine or destruction.

[0014] Accordingly, there is a need for methods to examine the genomes of potentially active biological pathogens in biological materials that have been subjected to sterilization in order to differentiate between biological pathogens that have been rendered inactive and those that are still potentially active. By doing so, such a method permits the determination of how effective a particular sterilization technique may be with respect to a particular biological pathogen. No presently available test provides such information.

[0015] PCR (polymerase chain reaction) is a method for increasing the concentration of a segment of a target sequence in a mixture of nucleic acid sequences without cloning or purification. (See K. B. Mullis et al., U.S. Pat. Nos. 4,683,195 and 4,683,202). This process for amplifying the target sequence consists of introducing two oligonucleotide primers to the sample containing the desired target nucleic acid sequence, followed by thermal cycling in the presence of a DNA polymerase. The two primers are complementary to their respective strands of the target sequence. To effect amplification, the genetic material within the sample is first denatured and then the primers are annealed to their complementary sequences within the target molecule. Following annealing, the primers are extended with a polymerase so as to form a new pair of complementary strands. The steps of denaturation, annealing and extension can be repeated many times (i.e., denaturation, annealing and extension constitute one “cycle”; there can be numerous “cycles”) to obtain a high concentration of an amplified segment of the desired target sequence. The length of the amplified segment of the desired target sequence is determined by the relative positions of the primers with respect to each other, and therefore, this length is a controllable parameter. Because the desired amplified segments of the target sequence become the predominant sequences (in terms of concentration) in the mixture, they are said to be “PCR amplified”. The segment of genetic material that has been amplified is generally referred to as an “amplicon”. The conditions employed for PCR reactions, including aspects of the timing, temperature(s) and particular polymerase selection are typically optimized for examining relatively short segments of nucleic acids, generally in the range of 50-200 nucleic acid residues. With PCR, it is possible to amplify a single copy of a specific target sequence in genomic DNA to a level detectable by several different methodologies (e.g., hybridization with a labelled probe; incorporation of biotinylated primers followed by avidin-enzyme conjugate detection; incorporation of ³²P-labelled deoxynucleotide triphosphates, e.g., dCTP or dATP, into the amplified segment). In addition to genomic DNA, any oligonucleotide sequence can be amplified with the appropriate set of primer molecules.

[0016] End-point PCR is a polynucleotide amplification protocol. The amplification factor that is observed is related to the number (n) of cycles that have occurred and the efficiency of replication at each cycle (E), which, in turn, is a function of the priming and extension efficiencies during each cycle. Amplification has been observed to follow the form E^(n), until high concentrations of the PCR product have been made.

[0017] At these high product concentrations, the efficiency of replication tends to drop significantly. It has been suggested that this is probably due to the displacement of the primers by the longer complementary strands of the PCR product. At concentrations in excess of 10⁻⁸ M, the rate of the two complementary PCR amplified product strands finding each other during the priming reactions becomes sufficiently fast that it may occur before or concomitantly with the extension step of the PCR process. This ultimately leads to a reduced priming efficiency, and, consequently, a reduced cycle efficiency. Continued cycles of PCR lead to declining increases of PCR product molecules, until the PCR product eventually reaches a plateau concentration (the “end-point”), usually a concentration of approximately 10⁻⁸ M. As a typical reaction volume is about 100 microlitets, this corresponds to a yield of about 6×10¹¹ double stranded product molecules.

[0018] Real-time PCR is also a polynucleotide amplification protocol, but PCR product analysis occurs simultaneously with amplification of the target sequence. Detecting agents, such as DNA dyes or fluorescent probes, can be added to the PCR mixture before amplification and used to analyze PCR products during amplification. Sample analysis occurs concurrently with amplification in the same tube within the same instrument. This combined approach decreases sample handling, saves time, and greatly reduces the risk of product contamination, as there is no need to remove the samples from their closed containers for further analysis. The concept of combining amplification with product analysis has become known as “real time” or “quantitative” PCR. (See, e.g., WO/9746707A2, WO/9746712A2 and WO/9746714A1).

[0019] Originally, monitoring fluorescence each cycle of PCR involved the use of ethidium bromide. See Higuchi et al., “Simultaneous amplification and detection of specific DNA sequences,” Bio/Technology 10:413-417 (1992); Higuchi et al., “Kinetic PCR analysis: real time monitoring of DNA amplification reactions,” Bio/Technology 11:1026-1030 (1993). In that system, fluorescence was measured once per cycle as a relative measure of product concentration. Ethidium bromide detects double stranded DNA; thus, if the desired target nucleic acid sequence is present, fluorescence intensity increases with temperature cycling (otherwise no fluorescence). Furthermore, the cycle number where an increase in fluorescence is first detected increases inversely proportionally to the log of the initial target sequence concentration. Other fluorescent systems have since been developed that are capable of providing additional data concerning the nucleic acid concentration.

[0020] In view of the difficulties and problems discussed above, there remains a need for a simple, yet accurate method of determining the efficiency of methods of sterilizing biological materials that act upon the genetic material of potentially active biological pathogens.

[0021] Each of the above references is incorporated by reference herein where appropriate for teachings of additional or alternative details, features and/or technical background.

SUMMARY OF THE INVENTION

[0022] An object of the invention is to solve at least the problems and/or disadvantages of the relevant art, and to provide at least the advantages described hereinafter.

[0023] Accordingly, it is an object of the present invention to provide methods of determining the level of potentially active biological pathogens in a biological material. Other objects, features and advantages of the present invention will be set forth in the detailed description of preferred embodiments that follows, and in part will be apparent from the description or may be learned by practice of the invention. These objects and advantages of the invention will be realized and attained by the compositions and methods particularly pointed out in the written description and claims hereof.

[0024] In accordance with these and other objects, a first embodiment of the present invention is directed to a method for determining the level of potentially active biological pathogens in a biological material, which comprises: (i) adding to a biological material an effective amount of at least two nucleic acid primer pairs, wherein a first nucleic acid primer pair hybridizes under stringent conditions to a first target nucleic acid sequence found in the biological pathogen and a second nucleic acid primer pair hybridizes under stringent conditions to a second target nucleic acid sequence found in the biological pathogen, and further wherein first and second target nucleic acid sequences are not identical and the second target nucleic acid sequence contains more nucleic acid residues than the first; (ii) amplifying the target nucleic acid sequences by polymerase chain reaction, which comprises adding at least one polymerase to the biological material containing the primer pairs to form an amplification mixture and thermally cycling this amplification mixture between at least one denaturation temperature and at least one elongation temperature for a period of time sufficient to amplify the target nucleic acid sequences; and (iii) detecting and quantifying the target nucleic acid sequences, wherein the quantity of the first target nucleic acid sequence is proportional to the number of biological pathogens in the biological material and the quantity of the second target nucleic acid sequence is proportional to the number of potentially active biological pathogens in the biological material.

[0025] Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention. The objects and advantages of the invention may be realized and attained as particularly pointed out in the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026]FIG. 1 shows the complete genomic nucleic acid sequence of human Parvovirus B19, and indicates exemplary sequences for preparing suitable forward and reverse primers and probes.

[0027]FIG. 2 shows the complete genomic nucleic acid sequence of hepatitis B virus, and indicates exemplary sequences for preparing suitable forward and reverse primers and probes.

[0028]FIG. 3 shows the complete genomic nucleic acid sequence of porcine Parvovirus, and indicates exemplary sequences for preparing suitable forward and reverse primers and probes.

[0029]FIG. 4 shows the complete genomic nucleic acid sequence of Sindbis virus, and indicates exemplary sequences for preparing suitable forward and reverse primers and probes.

[0030]FIG. 5 shows the complete genomic nucleic acid sequence of West Nile virus, and indicates exemplary sequences for preparing suitable forward and reverse primers and probes.

[0031]FIGS. 6A and 6B show the genomic nucleic acid sequence of the 16S ribosomal RNA gene and the 23S ribosomal RNA gene of Escherichia coli, and indicate exemplary sequences for preparing suitable forward and reverse primers and probes.

[0032]FIGS. 7A and 7B show the genomic nucleic acid sequence of the 18S ribosomal RNA gene and the 25S ribosomal RNA gene of yeast (S. cerevisiae), and indicate exemplary sequences for preparing suitable forward and reverse primers and probes.

[0033]FIG. 8 shows the complete nucleic acid sequence of human mitochondrial DNA, and indicates exemplary sequences for preparing suitable forward and reverse primers and probes.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0034] A. Definitions

[0035] Unless defined otherwise, all technical and scientific terms used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the relevant art.

[0036] As used herein, the singular forms “a,” “an,” and “the” include the plural reference unless the context clearly dictates otherwise.

[0037] As used herein, the term “biological material” is intended to mean any substance derived or obtained from a living organism. Illustrative examples of biological materials include, but are not limited to, the following: cells; tissues; blood or blood components; proteins, including recombinant and transgenic proteins, and proetinaceous materials; enzymes, including digestive enzymes, such as trypsin, chymotrypsin, alpha-galactosidase and iduronodate-2-sulfatase; immunoglobulins, including mono and polyimmunoglobulins; botanicals; food and the like. Preferred examples of biological materials include, but are not limited to, the following: ligaments; tendons; nerves; bone, including demineralized bone matrix, grafts, joints, femurs, femoral heads, etc.; teeth; skin grafts; bone marrow, including bone marrow cell suspensions, whole or processed; heart valves; cartilage; corneas; arteries and veins; organs, including organs for transplantation, such as hearts, livers, lungs, kidneys, intestines, pancreas, limbs and digits; lipids; carbohydrates; collagen, including native, afibrillar, atelomeric, soluble and insoluble, recombinant and transgenic, both native sequence and modified; chitin and its derivatives, including NO-carboxy chitosan (NOCC); stem cells, islet of Langerhans cells and other cells for transplantation, including genetically altered cells; red blood cells; white blood cells, including monocytes; and platelets. Additional examples of biological materials include forensic samples, human or animal remains, stomach contents, mummified remains of a once-living organism, fossilized remains, a product of manufacture containing or previously in contact with a biological material, and fomites.

[0038] As used herein, the term “biological pathogen” is intended to mean a biological pathogen that, upon direct or indirect contact with a biological material, may have a deleterious effect on the biological material or upon a recipient thereof. Such biological pathogens include, but are not limited to, the various viruses, bacteria (whether in the vegetative or spore state, including inter- and intracellular bacteria, such as mycoplasmas, ureaplasmas, nanobacteria, chlamydia, rickettsias), fungi (including yeasts) and/or single- or multi-cell parasites and pests known to those of skill in the art to generally be found in or infect biological materials.

[0039] Illustrative examples of some biological pathogens include, but are not limited to, the following: viruses, such as human immunodeficiency viruses and other retroviruses, herpes viruses, filoviruses, circoviruses, paramyxoviruses, cytomegaloviruses, hepatitis viruses (including hepatitis A, B, C, and D variants thereof, among others), pox viruses, toga viruses, Ebstein-Barr viruses and parvoviruses; bacteria, such as Escherichia, Bacillus, Campylobacter, Clostridium, Streptococcus and Staphylococcus; nanobacteria; single- and multi-cell parasites, such as Trypanosoma and malarial parasites, including Plasmodium species; fungi; yeasts; mycoplasmas and ureaplasmas; chlamydia; rickettsias, such as Coxiella burnetti; and multi-cell pests and the like.

[0040] Additional non-limiting examples of pathogens found in biological materials include the following bacteria: Escherichia, Bacillus, Campylobacter, Helicobacter, Lysteria, Clostridium, Streptococcus, Enterococcus, Staphylococcus, Brucella, Haemophilus, Salmonella, Yersinia, Pseudomonas, Serratia, Enterobacter, Kebsiella, Proteus, Citrobacter, Corynebacterium, Propionibacterium and Coxiella, such as Staphylococci (including, for example, S. aureus, S. epidermidis, S. saprophyticus, among others), Chlamydia (including, for example, C. pneumoniae, among others), Streptococci (including, for example, the viridians group of Streptococci: S. sanguis, S. oralis (mitis), S. salivatius, S. mutans, and others; and other species of Streptococci, such as S. bovis and S. pyogenes), Enterococci (for example, E. faecalis and E. faecium, among others), various fungi, and the AHACEK group of gram-negative bacilli (Haemophilus parainfluenzae, Haemophilus aphrophilus, Actinibacillus actnomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae), Neisseria gonorrhoeae, Clostridia sp., Listeria moncytogenes, Salmonella sp., Bacteroides fragilis, Escherichia coli, Proteus sp, and Klebsiella-Enterobacter-Serratia sp., among others.

[0041] Still other non-limiting examples of pathogens found in biological materials include the following viruses: Adeno-associated Virus (AAV), California Encephalitis Virus, Coronavirus, Coxsackievirus-A, Coxsackievirus-B, Eastern Equine Encephalitis Virus (EEEV), Echovirus, Hantavirus, Hepatitis A Virus (HAV), Hepatitis C Virus (HCV), Hepatitis Delta Virus (HDV), Hepatitis E Virus (HEV), Hepatitis G Virus (HGV), Human Immunodeficiency Virus (HIV), Human T-lymphotrophic Virus (HTLV), Influenza Virus (Flu Virus), Measles Virus (Rubeola), Mumps Virus, Norwalk Virus, Parainfluenza Virus, Polio virus, Rabies Virus, Respiratory Syncytial Virus, Rhinovirus, Rubella Virus, Saint Louis Encephalitis Virus, Western Equine Encephalitis Virus (WEEV), Yellow Fever Virus, Adenovirus, Cytomegalovirus (CMV), Epstein-Barr Virus (EBV), Hepatitis B Virus (HBV), Herpes Simplex Virus 1 (HHV1), Herpes Simplex Virus 2 (HHV2), Molluscum contagiosum, Papilloma Virus (HPV), Smallpox Virus (Variola), Vaccinia Virus, Venezuelan Equine Encephalitis Virus (VEEV), Ebola Virus, West Nile Virus, Human Parvovirus B19 and Rotavirus.

[0042] As used herein, the term “potentially active biological pathogen” is intended to mean a biological pathogen that is capable of causing a deleterious effect, either alone or in combination with another factor, such as a second biological contaminant or pathogen or a native protein (wild-type or mutant) or antibody, in the biological material and/or a recipient thereof.

[0043] As used herein, the term “wild-type” in reference to a nucleic acid sequence an amino acid sequence is intended to refer to the corresponding sequence found in naturally occurring organisms, such as biological pathogens, including such variants and mutants as are known to those skilled in the art.

[0044] As used herein, the term “sterilize” is intended to mean a reduction in the level of at least one potentially active biological pathogen found in the biological material being treated.

[0045] As used herein, the term “radiation” is intended to mean radiation of sufficient energy to sterilize at least some component of the irradiated biological material. Types of radiation include, but are not limited to, the following: (i) corpuscular (streams of subatomic particles such as neutrons, electrons, and/or protons); (ii) electromagnetic (originating in a varying electromagnetic field, such as radio waves, visible (both mono and polychromatic) and invisible light, infrared, ultraviolet radiation, x-radiation, and gamma rays and mixtures thereof); and (iii) sound and pressure waves. Such radiation is often described as either ionizing (capable of producing ions in irradiated materials) radiation, such as gamma rays, and non-ionizing radiation, such as visible light. The sources of such radiation may vary and, in general, the selection of a specific source of radiation is not critical provided that sufficient radiation is given in an appropriate time and at an appropriate rate to effect sterilization. In practice, gamma radiation is usually produced by isotopes of Cobalt or Cesium, while UV and X-rays are produced by machines that emit UV and X-radiation, respectively, and electrons are often used to sterilize materials in a method known as “E-beam” irradiation that involves their production via a machine. Visible light, both mono- and polychromatic, is produced by machines and may, in practice, be combined with invisible light, such as infrared and UV, that is produced by the same machine or a different machine.

[0046] B. Particularly Preferred Embodiments

[0047] A first preferred embodiment of the present invention is directed to a method for determining the level of potentially active biological pathogens in a biological material, which comprises:

[0048] (i) adding to a biological material an effective amount of at least two nucleic acid primer pairs,

[0049] wherein a first nucleic acid primer pair hybridizes under stringent conditions to a first target nucleic acid sequence found in the biological pathogen and a second nucleic acid primer pair hybridizes under stringent conditions to a second target nucleic acid sequence found in the biological pathogen, and further wherein first and second target nucleic acid sequences are not identical and the second target nucleic acid sequence contains more nucleic acid residues than the first;

[0050] (ii) amplifying the target nucleic acid sequences by polymerase chain reaction, which comprises adding at least one polymerase to the biological material containing the primer pairs to form an amplification mixture and thermally cycling this amplification mixture between at least one denaturation temperature and at least one elongation temperature for a period of time sufficient to amplify the target nucleic acid sequences; and

[0051] (iii) detecting and quantifying the target nucleic acid sequences, wherein the quantity of the first target nucleic acid sequence is proportional to the number of biological pathogens in the biological material and the quantity of the second target nucleic acid sequence is proportional to the number of potentially active biological pathogens in the biological material.

[0052] The first and second target nucleic acid sequences employed in the methods of the present invention are preferably selected to be specific for a particular biological pathogen of interest. That is, it is preferred that at least one, and more preferably both, of the first and second nucleic acid sequences is found only in the biological pathogen of interest and not in any other component of the biological material. According to these embodiments of the present invention, such a selection (or selections) for the target nucleic acid sequence(s) allows for the selective determination of the levels of biological pathogen, including the total number of biological pathogens present (potentially active and inactive) as well as the number of potentially active pathogens and the number of inactive pathogens.

[0053] One skilled in the art may determine suitable target nucleic acid sequences empirically, based on factors such as the particular biological pathogen(s) of interest, the biological material being tested and the PCR conditions selected.

[0054] Preferably, the first target nucleic acid sequence contains between about 50 and about 500 nucleic acid residues. More preferably, the first target nucleic acid sequence contains between about 50 and about 250 nucleic acid residues, and most preferably between about 50 and about 150 nucleic acid residues.

[0055] The second target nucleic acid sequence preferably contains between about 500 and about 50,000 nucleic acid residues. More preferably, the second target nucleic acid sequence contains between about 1000 and about 10,000 nucleic acid residues, even more preferably between about 2000 and about 5000 nucleic acid residues and most preferably between about 2500 and about 5000 nucleic acid residues.

[0056] The first and second target nucleic acid sequences may be completely different or they may overlap by some or all of the shorter of the two. According to certain preferred embodiments of the present invention, the first target nucleic acid sequence and the second nucleic acid sequence contain at least 16 contiguous nucleic acid residues in common.

[0057] As noted, the first and second target nucleic acid sequences are preferably selected to be specific for a biological pathogen of interest. According to such embodiments of the present invention, the biological pathogen is preferably selected from the group consisting of bacteria, viruses, mycoplasmas, fungi and single cell parasites.

[0058] According to these embodiments of the present invention, at least one of the first and second target nucleic acid sequences, and more preferably both the first and second target nucleic acid sequence, are at least 30% homologous to a wild-type nucleic acid sequence found in the biological pathogen of interest. More preferably, the first and/or second target nucleic acid sequence is at least 50% homologous to a wild-type nucleic acid sequence found in the biological pathogen of interest, and even more preferably at least 70% homologous. Most preferably, the first and/or second target nucleic acid sequence is at least 90% homologous to a wild-type nucleic acid sequence found in the biological pathogen of interest. According to certain preferred embodiments, the first and/or second target nucleic acid sequence is substantially identical to a wild-type nucleic acid sequence found in the biological pathogen of interest.

[0059] According to particularly preferred embodiments of the present invention, at least one of the first and second target nucleic acid sequences, and more preferably both, is a nucleic sequence that is highly conserved among different species, different genera or even different families of biological pathogens.

[0060] For example, if the biological pathogen of interest is bacteria, then the first and/or second target nucleic acid sequences are preferably sequences that are found in the gene encoding the 16S ribosomal RNA or the gene encoding the 23S ribosomal RNA. According to these preferred embodiments of the present invention, the first target nucleic acid sequence is even more preferably a nucleic acid sequence found in the gene encoding the 16S ribosomal RNA of bacteria. Preferably, such a sequence is conserved among different species and genera of bacteria.

[0061] Thus, as shown in FIG. 6A, suitable primers and probes were prepared from the gene encoding the 16S ribosomal RNA of bacteria that were useful for a wide range of bacterial biological pathogens, including Escerichia coli, Bacteroides forsythus, Porphyromonas gingivalis, Prevotella melaninogenica, Cytophaga baltica, Campylobacter jejuni, Helicobacter pylori, Trepnema denticola, Treponema pallidum, Leptothrix mobilis, Thiomicrospira dentrificans, Neisseria meningitides, Actinobacillus actinomycetemcomitans, Haemophilus influenzae, Salmonella typhi, Vibrio cholerae, Coxiella burnettti, Legionella pneumophila, Pseudomonas aeruginosa, Caulobacter vibrioides, Rhodospirillum rubrum, Nitrobacter winogradskyi, Wolbachia sp., Myxococcus xanthus, Corynebacterium diptheriae, Mycobacterium tuberculosis, Streptomyces coelicolor, Actinomyces odontolyticus, Bacillus subtilis, Staphylococcus aureus, Listeria monocytogenes, Enterococcus faecais, Lactobacillus acidophilus, Streptococcus mutans, Clostridium botulinum, Peptostreptococcus micros, Veillonella dispar, Fusobacterium nucleatum, Clamydia trachomatis, Mycoplasma pneumoniae.

[0062] According to these particularly preferred embodiments of the present invention, i.e. if the biological pathogen of interest is bacteria, then the second target nucleic acid sequence is preferably a nucleic acid sequence found in both the gene encoding the 16S ribosomal RNA and the gene encoding the 23S ribosomal RNA. According to these embodiments, the second target nucleic acid sequence is even more preferably a nucleic acid sequence found in the gene encoding the 16S ribosomal RNA and at least a portion of the gene encoding the 23S ribosomal RNA, as well as the non-coding nucleic sequence found therebetween in bacterial genomes.

[0063] Similarly, if the biological pathogen of interest is fungi, then the first and/or second target nucleic acid sequences are preferably sequences that are found in the gene encoding the 18S ribosomal RNA or the gene encoding the 25S ribosomal RNA. According to these embodiments of the present invention, the first target nucleic acid sequence is even more preferably a nucleic acid sequence found in the gene encoding the 18S ribosomal RNA of fungi. Preferably, such a sequence is conserved among different species and genera of fungi.

[0064] According to these particularly preferred embodiments of the present invention, i.e. if the biological pathogen of interest is fungi, then the second target nucleic acid sequence is preferably a nucleic acid sequence found in both the gene encoding the 18S ribosomal RNA and the gene encoding the 25S ribosomal RNA. According to these embodiments, the second target nucleic acid sequence is even more preferably a nucleic acid sequence found in the gene encoding the 18S ribosomal RNA and at least a portion of the gene encoding the 25S ribosomal RNA and the non-coding nucleic sequence found therebetween in fungal genomes, and most preferably a nucleic acid sequence found in the gene encoding the 18S ribosomal RNA, at least a portion of the gene encoding the 25S ribosomal RNA and the gene encoding the 5.8S ribosomal RNA, as well as both non-coding nucleic sequences found therebetween in fungal genomes.

[0065] The first and second pairs of nucleic acid primers are each selected based on their ability to generate the desired target nucleic acid sequences under the appropriate PCR conditions. Accordingly, each primer must be specific for the desired target nucleic acid sequence. Similarly, each primer must be selected so that they are not self-complementary or complementary to another primer (or probe, if present).

[0066] According to certain preferred embodiments of the present invention, at least one member of each pair of nucleic acid primers is substantially identical, i.e. one of the first pair of nucleic acid primers and one of the second pair of nucleic acid primers are substantially identical.

[0067] According to other preferred embodiments of the present invention, the two pairs of nucleic acid primers are completely different, i.e., neither of the first pair of nucleic acid primers is substantially identical to either of the second pair of nucleic acid primers.

[0068] According to still other preferred embodiments of the present invention, the two pairs of nucleic acid primers are substantially identical, i.e. one of the first pair of nucleic acid primers is substantially identical to one of the second pair of nucleic acid primers and the other one of the first pair is identical to the other one of the second pair. According to such embodiments, two distinct target sequences may still be obtained, for example, in the case where one or both members of each primer pair hybridize to more than one sequence, for example, as in the case where the first and second target sequences ate part of a circular nucleic acid sequence, such as a plasmid, where the hybridization location of the primers on the circular nucleic acid sequence is such that transcription in different directions leads to two different amplicons. Similarly, in cases where the first and target sequences are highly homologous, particularly at their respective 5′ and 3′ ends, then the primers will hybridize to both, such that transcription leads to two different amplicons.

[0069] The polymerize chain reaction employed in the inventive methods is performed according to the methods and techniques known to those skilled in the art, i.e., a nucleic acid primer pair is added to the biological material containing the sequence of interest to form an amplification mixture that is then thermally cycled for a sufficient period of time to amplify the desired sequence. The thermal cycling generally comprises cycling the amplification mixture between at least one denaturation temperature and at least one elongation temperature. Preferably, the thermal cycling comprises cycling the amplification mixture between at least one denaturation temperature, at least one annealing temperature and at least one elongation temperature.

[0070] Specific temperatures for use in denaturation, elongation and/or annealing may be determined empirically by one skilled in the art based, for example, on the specific target sequence being amplified and the particular probes employed. Likewise, the specific time(s) that the amplification mixture is maintained at the various denaturation, elongation and/or annealing temperature(s) may be determined empirically by one skilled in the art based on similar considerations.

[0071] According to particularly preferred embodiments of the present invention, the elongation temperature selected for use in the PCR of the inventive methods is not more than about 70° C. More preferably, the elongation temperature selected is between about 60° C. and about 69° C., and even more preferably between about 65° C. and about 69° C. Most preferably, the elongation temperature employed in the PCR of the inventive methods is about 68° C.

[0072] According to additional preferred embodiments of the present invention, the denaturation temperature selected for use in the PCR of the inventive methods is not more than about 95° C. More preferably, the denaturation temperature selected is between about 90° C. and about 95° C., and even more preferably between about 92° C. and about 95° C. Most preferably, the denaturation temperature employed in the PCR of the inventive methods is about 94° C.

[0073] According to other preferred embodiments of the present invention, when the thermal cycling includes an annealing temperature, the annealing temperature selected is about 5-10° C. below the melting temperature of the primers being employed. Preferably, the annealing temperature selected is not more than about 65° C. More preferably, the annealing temperature selected is between about 57° C. and about 63° C., and even more preferably between about 58° C. and about 62° C. Most preferably, the annealing temperature employed in the PCR of the inventive methods is about 60° C.

[0074] According to additional preferred embodiments of the present invention, during each thermal cycle, the amplification mixture is maintained at the elongation temperature for a period of not less than about 1 minute. More preferably, during each thermal cycle, the amplification mixture is maintained at the elongation temperature for a period of not less than about 2 minutes, and even more preferably for a period of not less than about 3 minutes.

[0075] According to particularly preferred embodiments of the present invention, the amplification mixture is maintained at the elongation temperature for a period of not less than about 2 minutes during the first cycle of the thermal cycling, and then the period during which said amplification mixture is maintained at the elongation temperature is increased by a period of about 5 seconds for each successive thermal cycle. Thus, for example, according to such embodiments of the present invention, if the amplification mixture was maintained at the elongation temperature for a period of 2 minutes during the first cycle of the thermal cycling, it would be maintained at the elongation temperature for a period of 2 minutes, 5 seconds for the second cycle, 2 minutes, 10 seconds for the third cycle, 2 minutes, 15 seconds for the fourth cycle, and so on until the thermal cycling is completed.

[0076] According to additional preferred embodiments of the present invention, during each thermal cycle, the amplification mixture is maintained at the denaturation temperature for a period of not more than about 1 minute. More preferably, during each thermal cycle, the amplification mixture is maintained at the denaturation temperature for a period of not more than about 45 seconds, and even more preferably for a period of not more than about 30 seconds, and still even more preferably for a period of not more than about 20 seconds. Most preferably, during each thermal cycle, the amplification mixture is maintained at the denaturation temperature for a period of not more than about 15 seconds, such as a period of about 10 seconds.

[0077] According to still other preferred embodiments of the present invention, when the thermal cycling includes an annealing temperature, the amplification mixture is maintained at the annealing temperature for a period of not less than about 30 seconds. More preferably, according to such embodiments, during each thermal cycle, the amplification mixture is maintained at the annealing temperature for a period between 30 seconds and 2 minutes, and even more preferably for a period of not less than about 45 seconds. Most preferably, during each thermal cycle, the amplification mixture is maintained at the annealing temperature for a period of about 1 minute.

[0078] The number of thermal cycles employed in the PCR of the inventive methods may be determined empirically by one skilled in the art depending, for example, on the suspected concentration of the target sequence of interest in the biological material being tested. According to preferred embodiments of the present invention, the amplification mixture is subjected to at least about 30 cycles of thermal cycling, and even more preferably at least about 40 cycles. Most preferably, the amplification mixture is subjected to at least about 50 cycles of thermal cycling.

[0079] The polymerase employed in the PCR of the inventive methods may be any of the suitable polymerases known to those skilled in the art. Preferably, the polymerase employed is a thermostable polymerase, i.e. a polymerase that is not adversely affected by the higher temperatures involved in thermal cycling. More preferably, the polymerase may be a Taq polymerase, or a suitable derivative thereof and/or a proof-reading polymerase.

[0080] According to particularly preferred embodiments of the present invention, at least two polymerases are employed in the PCR of the inventive methods. Preferably, at least one of the polymerases is a Taq polymerase or a suitable derivative thereof, such as TaqMan DNA polymerase (available from Applied BioSystems), and the other polymerase is a proof-reading polymerase, such as ProofStart DNA polymerase (available from Qiagen).

[0081] According to certain preferred embodiments of the present invention, the amplification mixture further contains at least one thermostable inorganic pyrophosphatase. Suitable amounts of thermostable inorganic pyrophosphatase may be determined empirically by one skilled in art. Generally, when present, the ratio of thermostable inorganic pyrophosphatase to Taq polymerase is at least about 1:20, more preferably at least about 1:10 and even more preferably at least about 1:5.

[0082] The remaining parameters employed in the PCR of the inventive methods, such as the primer concentration (generally about 100-500 nM and preferably about 200 nM)), magnesium concentration (generally 1.5-6 mM and preferably about 1.5 mM of magnesium sulfate and/or magnesium chloride), deoxyribonucleotide triphosphates (dNTP) concentration (generally about 0.2-0.4 mM each and preferably about 0.2 mM each), probe concentration (if present, generally about 50-800 nM, and preferably about 100 nM), may each be determined empirically by one skilled in the art using any of the known methods and techniques.

[0083] According to certain particularly preferred embodiments of the present invention, the deoxyribonucleotide ttiphosphates (dNTP) that are employed in the PCR of the inventive methods are selected from the group consisting of C, T, G and A. Preferably, substantially no dUTP is present in the amplification mixture of the inventive methods. According to still further preferred embodiments, substantially no uracil N-glycosylase is present in the amplification mixture of the inventive methods.

[0084] According to certain particularly preferred embodiments of the present invention, the amplification mixture further comprises at least one buffer solution. Suitable buffer solutions are known and available to those skilled in the art. Particularly preferred buffer solutions include pH modifying buffers, such as buffers containing Tris-HCl, and buffers which maintain salt concentration, particular magnesium concentration, such as buffers containing KCl and/or (NH₄)₂SO₄.

[0085] After amplification using PCR, the first and second target nucleic acid sequences are detected and quantified. This detecting and quantifying may be conducted using any of the methods and techniques known to those skilled in the art. For example, detecting and quantifying of the first and second nucleic acid sequences may be conducted by adding a suitable detecting agent, such as an intercalating dye, directly to the amplification mixture or by adding a suitable nucleic acid probe to the mixture, preferably either a suitable nucleic acid probe in combination with a detecting agent or a suitable nucleic acid probe having a detectable label covalently or ionically attached thereto or complexed therewith.

[0086] Preferably, the first and second target nucleic acid sequences are detected by adding at least one nucleic acid probe to the biological material being tested. If the first and second target nucleic acid sequences were amplified in a single reaction vessel, then it is preferable to use at least two nucleic acid probes, one of which is specific for the first target nucleic acid sequence and the other of which is specific for the second target nucleic acid sequence. Conversely, if the first and second target nucleic acid sequences were amplified in separate reaction vessels, then the same nucleic acid probe may be used for detecting both the first target nucleic acid sequence and the second target nucleic acid sequence.

[0087] Any nucleic acid probe employed in the inventive methods should contain sufficient nucleic acid residues to hybridizes selectively under stringent conditions to a specific desired nucleic acid sequence, i.e. suitable probes will generally contain at least 16 nucleic acid residues, and preferably hybridizes selectively under stringent conditions to a specific nucleic acid sequence of the first and/or second target nucleic acid sequence that is not the same as the nucleic acid sequence of any of the primers. Suitable nucleic acid probes include, but are not limited to, 5′ nuclease probes, hairpin probes, adjacent probes, sunrise probes and scorpion probes.

[0088] According to certain preferred embodiments of the present invention, the nucleic acid probe employed in the inventive methods has an endogneous passive dye, such as Tamra or the like. In other preferred embodiments, such an endogenous passive dye may be replaced by a passive dye that is not covalently bound to the probe, such as Rox or the like.

[0089] According to certain particularly preferred embodiments of the present invention, prior to step (i), the biological material being tested has been subjected to a process that alters at least one wild-type nucleic acid sequence in the biological pathogen of interest. Such processes may cause the wild-type nucleic acid sequence to break, cross-link and/or complex. An illustrative, but non-limiting, example of such a process is irradiation of the biological material with ionizing radiation, such as UV or gamma radiation.

[0090] Althogh not limited in application, the inventive methods are particularly useful in determining the effectiveness of processes that alter nucleic acid sequences, such as the inactivation of biological pathogens by gamma irradiation. More specifically, conventional PCR testing methods only determine whether a particular biological pathogen is present in a biological material, not whether that biological pathogen is active or inactive. The methods of the present invention, however, may be used to determine not only whether a particular biological pathogen is present in a biological material as shown by amplification of the first target sequence, but also whether that biological pathogen is inactive by virtue of an altered wild-type nucleic acid sequence as shown by a relative delay in the amplification of the second target sequence (the greater the delay in amplification, the greater the reduction in the level of potentially active biological pathogens). Thus, the inventive methods are useful for evaluating the effectiveness of sterilization processes because they determine both the original level and the residual level of potentially active biological pathogens.

EXAMPLES

[0091] The following examples are illustrative, but not limiting, of the present invention. Other suitable modifications and adaptations are of the variety normally encountered by those skilled in the art and are fully within the spirit and scope of the present invention.

Example 1

[0092] Purpose: To demonstrate linear amplification of B19 DNA.

[0093] Materials: 1. B19 virus, titer 7.6×10¹¹ iu/ml from Bayer;

[0094] 2. SNAP whole blood DNA isolation kit;

[0095] 3. Forward Primer: Prism 5 (FIG. 1);

[0096] 4. Reverse Primer: Prism 6 (FIG. 1);

[0097] 5. Probe 3 (FIG. 1) labeled with FAM at 5′ end and TAMRA at 3′ end;

[0098] 6. TaqMan Universal Master Mix, (ABI; cat. no. 4304437);

[0099] 7. DNASE, RNASE free water;

[0100] 8. ABI 96 well plate and adhesive cores;

[0101] 9. ANI 7000.

[0102] Procedure: 1. Followed SNAP protocol for extraction of 100 μL B19 sample, eluted in 100 μl TE;

[0103] 2. Diluted primers to 18 μM with TE;

[0104] 3. Diluted probe to 5 μM with TE;

[0105] 4. Prepared the following master mix: TaqMan Master Mix:   25 μl; Prism 5:  2.5 μl; Prism 6:  2.5 μl; Taqman Probe  2.5 μl; Water: 12.54 μl;

[0106] 5. Added 45 μl of master mix per well;

[0107] 6. Serially diluted B19 DNA, adding water to the NTC well;

[0108] 7. Sealed and centrifuged the plate at 2300 rpm for about 30 seconds;

[0109] 8. Ran PCR program for 50 cycles.

[0110] Results: A standard dilution curve was observed for B19 infected plasma, validating primer pair Prism 5 and Prism 6 with Probe 3.

Example 2

[0111] Purpose: To examine irradiated and unirradiated samples containing PPV using a 549 bp amplicon.

[0112] Materials: 1. PPV (irradiated at 0 kGy, 50 kGy, 65 kGy, 75 kGy or 85 kGy);

[0113] 2. SNAP Protein Degrader;

[0114] 3. Cell Lysis Buffer;

[0115] 4. Tris-HCl;

[0116] 5. Primers: Prism 11 and Prism 12 (FIG. 3); and

[0117] 6. Probe 6 (FIG. 3).

[0118] Procedure: 1. To 100 μl viral sample, added 50 μl tris-HCl buffer, 60 μl protein degrader, and 200 μl cell lysis buffer;

[0119] 2. Mixed and incubated for 25 minutes (5 minutes at 70° C.);

[0120] 3. Diluted samples to 1/50, 1/500, 1/5000, 1/25000, 1/50000, 1/250000 and 1/500000;

[0121] 4. Ran PCR for 55 cycles.

[0122] Results: Results showed that unirradiated material had regular dilution series curves, irradiated material (50 kGy) behaved differently, dilute material did not amplify showing a reduction in the number of copies of the target sequence.

Example 3

[0123] Purpose: To determine effects of gamma irradiation (0 kGy sample, 50 kGy sample, mixture of 0+50 kGy sample and 75 kGy sample) on samples containing PPV analyzed by PCR.

[0124] Materials: 1. PPV (irradiated at 0 kGy, 50 kGy or 75 kGy);

[0125] 2. Primers: Prism 11 & Prism 12, Probe 6 (FIG. 3);

[0126] 3. Primers: Prism 1 & Prism 2, Probe 1 (FIG. 3).

[0127] Procedure: 1. Diluted samples containing PPV to 1/100, 1/1000, 1-2000, 1/10000, 1/20000, 1/40000 and 1/400000 (0 kGy, 50 kGy, 0+50 kGy and 75 kGy); 2. Ran PCR program for 55 cycles.

[0128] Results: Irradiaition to 50 kGy of PPV material reduced amplification of 549 bp amplicon.

Example 4

[0129] Purpose: To examine the relative effectiveness of Qiagen and Taqman reagents on samples containing PPV.

[0130] Materials: 1. PPV DNA (phenol extracted);

[0131] 2. Taq PCR Core Kit;

[0132] 3. ProofStart DNA polymerase;

[0133] 4. Taqman Universal PCT Master Mix;

[0134] 5. Prism 1, 2, 11 and 17 (FIG. 3);

[0135] 6. Probes 1 and 6 (FIG. 3);

[0136] 7. Agarose;

[0137] 8. TAE;

[0138] 9. EtBr.

[0139] Procedure: 1. Prepared the following four master mixes: a. Qiagen: 1 2 10x buffer:   30 μl   25 μl dNTP's:    9 μl  7.5 μl pA:  8.34 μl 6.95 μl pB:  8.34 μl 6.95 μl taq:    6 μl   5 μl H₂O: 187.32 μl  156.1 μl  probe:   15 μl 12.5 μl b. Taqman: 3 4 Master Mix:   150 μl  125 μl pA:   15 μl 12.5 μl pB:   15 μl 12.5 μl probe:   15 μl 12.5 μl H₂O:   69 μl 57.5 μl

[0140] 2. Pipetted 44 μl of master mix 1 into row D, wells 1 and 2; row E, wells 1 and 2; and row H, well 1, of a well plate;

[0141] 3. Pipetted 44 μl of master mix 2 into row D, wells 3 and 4; and row E, wells 3 and 4, of a well plate;

[0142] 4. Pipetted 44 μl of master mix 3, into row F, wells 1 and 2; row G, wells 1 and 2; and row H, well 3, of a well plate;

[0143] 5. Pipetted 44 μl of master mix 4 into row F, wells 3 and 4; and row G, wells 3 and 4, of a well plate;

[0144] 6. Added 1 μl of ProofStart taq to row D, wells 1-4 and row F, wells 1-4 and added 1 μl water to remaining wells;

[0145] 7. Added 5 μl water to row H, wells 1 and 3 and added 5 μl PPV DNA to remaining wells;

[0146] 8. Ran PCR for 40 cycles.

[0147] Results: Qiaqen Master with ProofStart taq produced functional large amplicons in realtime PCR with PPV DNA more efficiently than the TaqMan master mix.

Example 5

[0148] Purpose: To examine the effects of proofstart in amplifying large amplicons and to examine the effects of 50 kGy irradiation on PPV.

[0149] Materials: 1. PPV DNA (irradiated to 0 kGy and 50 kGy);

[0150] 2. Taq PCR Core Kit;

[0151] 3. Proofstart DNA polymerase;

[0152] 4. Prism 11, 16 and 17 (FIG. 3);

[0153] 5. Agarose;

[0154] 6. Ethidium Bromide;

[0155] 7. TAE buffer.

[0156] Procedure: 1. Set up PCR master mix as follows: 10x buffer: 50 μl dNTP's: 15 μl pA: 13.9 μl (primer 11) taq: 10 μl water: 347.2 μl

[0157] 2. Placed aliquots into PCR tubes;

[0158] 3. Added either primer 16 or 17 to PCR tubes;

[0159] 4. Added PPV DNA (diluted to 1:100) to each PCR tube:

[0160] 5. Added 10 μl proofstart to half of the samples (2 at 0 kGy and 2 at 50 kGy);

[0161] 6. Performed PCR (about 55 cycles)

[0162] 7. Poured a 1% gel and ran at 100 V for 20 minutes.

[0163] Results: Addition of a proofreading polymerase resulted in improved amplication of longer amplicons. Delay in amplification of target sequence in irradiated samples is proportional to damage done to viral genetic material.

Example 6

[0164] Purpose: To examine the effect of TSP concentration on amplification of large target amplicons in gamma irradiated and unirtadiated PPV.

[0165] Materials: 1. TSP (cat. no. M02965);

[0166] 2. Qiagen Core kit;

[0167] 3. ProofStart DNA polymerase;

[0168] 4. PPV (irradiated to 0 kGy or 50 kGy).

[0169] Procedure: 1. Prepared a master mix (standard PCR set-up) for each (TSP: Taq 1:20, 1:10, 1:5);

[0170] 2. Added 43.61 μl of each master mix (TSP titration) to PCR tubes;

[0171] 3. Added 1.39 μl of primers 16, 17 or 19 (FIG. 3) to appropriate PCT tubes;

[0172] 4. Added 5 μl water to the negative control, which contained primer pair 11, 16 (FIG. 3).

[0173] 5. Diluted PPV 1:100;

[0174] 6. Added PPV to PCR tubes;

[0175] 7. Performed PCR;

[0176] 8. Poured a 1% gel and ran at 100 V for 20 minutes.

[0177] Results: Under these conditions, addition of TSP resulted in increased amplification of target amplicons in both irradiated and unirradiated samples, but irradiation of PPV resulted in decreased amplification of target amplicon.

Example 7

[0178] Purpose: To examine the effects of gamma irradiation on amplification of PPV target amplicons of various sizes.

[0179] Materials: 1. PPV DNA (irradiated to 0 kGy or 50 kGy);

[0180] 2. Taq PCR Core Kit;

[0181] 3. ProofStart DNA Polymerase;

[0182] 4. Prism 11, 16, 17, 18 and 19 (FIG. 3);

[0183] 5. Agarose;

[0184] 6. TAE;

[0185] 7. Ethidium Bromide.

[0186] Procedure: 1. Prepared PCR Master Mix as follows: 10x Buffer   5 μl dNTPs  1.5 μl pA 1.39 μl pB 1.39 μl taq   1 μl water 33.72 μl  PPV   5 μl.

[0187] 2. Alliquoted samples into PCR tubes;

[0188] 3. Ran PCR;

[0189] 4. Poured a 1% agarose gel and ran at 120 V for about 1.5 hours.

[0190] Results: Irradiation to 50 kGy resulted in decreased amplification of larger target amplicons.

Example 8

[0191] Purpose: To examine PCR sensitivity and determine log reduction of PPV in samples irradiated to 50 kGy and having a starting concentration of 2.5×10⁷ gEq.

[0192] Materials: 1. Standard PCR reagents (Qiacen Core Kit, TSP, Proofstart, etc.);

[0193] 2. Primers 11 and 17 (FIG. 3);

[0194] 3. PPV extract.

[0195] Procedure: 1. Prepared master mix with primers 11 and 17;

[0196] 2. Performed a 10 fold dilution series from 10⁷ to 10⁰ of PPV extract;

[0197] 3. Pipetted 45 μl of master mix into PCR tubes;

[0198] 4. Pipetted 5 μl of each PPV dilution into appropriated PCR tubes;

[0199] 5. Added 5 μl water to control;

[0200] 6. Ran PCR;

[0201] 7. Ran samples in 1% agarose at 100V for about 47 minutes.

[0202] Results: Irradiation of sample to 50 kGy resulted in decreased amplification of target amplicon across all concentration ranges.

Example 9

[0203] Purpose: To examine PCR sensitivity and determine log reduction of PPV in samples irradiated to 50 kGy and having a starting concentration of 2.5×10⁷ gEq.

[0204] Materials: 1. TSP;

[0205] 2. Standard PCR kit (Qiacen with ProofStart Polymerase);

[0206] 3. Primers 11 and 19 (FIG. 3);

[0207] 4. PPV Extract (Irradiated to 0 kGy and 50 kGy).

[0208] Procedure: 1. Prepared master mix with primers 11 and 19;

[0209] 2. Performed a 10 fold dilution series from 10⁷ to 10⁰ of PPV extract;

[0210] 3. Pipetted 45 μl of master mix into PCR tubes;

[0211] 4. Pipetted 5 μl of each PPV dilution into appropriated PCR tubes;

[0212] 5. Added 5 μl water to control;

[0213] 6. Ran PCR as follows: 95° C. for 2 minutes (1 cycles)

[0214] 94° C. for 10 seconds (40 cycles)

[0215] 60° C. for 1 minute (40 cycles)

[0216] 68° C. for 2 minutes (40 cycles);

[0217] 7. Cooled to 4° C.;

[0218] 8. Ran samples on 1% agarose gel in 1×TAE and 5 μl/100 ml ethidium bromide at 100 V for 52 minutes (5 μl on gel).

[0219] Results: Irradiation to 50 kGy resulted in decreased amplification of target amplicon across all concentration ranges. For unirradiated samples, relative band strength of observed target amplicon decreased with decreasing concentration.

Example 10

[0220] Purpose: Primer validation for B19 using probe 7 and various primers.

[0221] Materials: 1. B19 IGIV Paste (irradiated to 0 kGY or 50 kGy);

[0222] 2. EXB;

[0223] 3. Proteinase;

[0224] 4. yeast tRNA

[0225] 5. phenol chloroform isoamyl alcohol;

[0226] 6. 3M NaAc;

[0227] 7. isopropanol;

[0228] 8.70% EtOH;

[0229] 9. TE buffer;

[0230] 10. Prisms 5, 6, 20, 21, 22, 23, 24, 25, 26 (FIG. 1);

[0231] 11. Qiagen reagents;

[0232] 12. Ampligold Taq;

[0233] 13. ProofStart Polymerase;

[0234] 14. Agarose;

[0235] 15. TAE;

[0236] 16. Ethidium Bromide.

[0237] Procedure: 1. Prepared a Master Mix as follows: Buffer 5 μl DNTP 1.5 μl   Taq 1 μl DNA 5 μl water 34.72 μl   

[0238] 2. Pipetted Master Mix into PCR tubes;

[0239] 3. Added the following primer pairs to appropriate PCR tubes: 20&21; 20&22; 20&23; 20&24; 20&25; 20&6; 20&26; 5&6;

[0240] 4. Ran PCR;

[0241] 5. ran 1% gel for about 1 hour.

[0242] Results: All tested primers yielded desired target amplicons.

Example 11

[0243] Purpose: Use of PCR multiplexing with target amplicons of about 112 bp and about 2.4 kbp for B19 virus in samples irradiated to 0 kGy or 50 kGy.

[0244] Materials: 1. TSP thermostable inorganic pyrophosphatase

[0245] 2. Standard PCR reagents;

[0246] 3. B19 viral extract (irradiated to 0 kGy and 50 kGy);

[0247] 4. Prisms 5, 6, 20 and 25 (FIG. 1);

[0248] 5. Taq;

[0249] 6. ProofStart Polymerase.

[0250] Procedure: 1. Prepared standard PCR set-up with 3× master mixes, for each primer set (primer sets: 5&6; 20&25; 5&6; and 20&25);

[0251] 2. Prepared appropriate PCR tubes containing the following primer pairs: (5, 6) 0 kGy; (5, 6) 50 kGy; (20, 25) 0 kGy; (20, 25) 50 kGy; (5, 6) & (20, 25), 0 kGy; and (5, 6) and (20, 25), 50 kGy;

[0252] 3. Added 5 μl B19 to PCR tubes containing 45 μl of appropriate master mix;

[0253] 4. Added 5 μl water to control;

[0254] 5. Ran PCR.

[0255] 6. Ran samples on 1% aragose gel at 100 V for about 17 minutes.

[0256] Results: PCR multiplexing is effective for mixtures containing large target amplicons and small target amplicons. Irradiation to 50 kGy resulted in decreased amplification of the large target amplicon relative to the small target amplicon.

Example 12

[0257] Purpose: Irradiated and unirtadiated samples containing B19 viral material were examined using real time PCR.

[0258] Materials: 1. B19 viral material (irradiated to 0 kGy and 50 kGy);

[0259] 2. Prism pairs (20, 21) and (20, 26) (FIG. 1);

[0260] 3. Qiagen PCR reagents;

[0261] 4. Qiagen ProofStart;

[0262] 5. Agarose;

[0263] 6. TAE (1×);

[0264] 7. sample loading buffer (SLB).

[0265] Procedure: 1. Prepared standard samples containing primer pairs with 10¹¹ to 10¹ dilution series;

[0266] 2. Ran PCR (40 cycles);

[0267] 3. Ran gel on 1% agarose (8 μl PCR product, 1 μl SLB) at 100 V for about 20 minutes.

[0268] Results: Unirradiated and irradiated samples amplified in a regular pattern for a dilution series with a small amplicon. As amplicon size increased, unirradiated material maintained a regular dilution pattern while irradiated material did not.

Example 13

[0269] Purpose: To investigate the effect of gamma irradiation on samples containing HBV clone and irradiated to 50 kGy.

[0270] Materials: 1. HBV (irradiated to 0 kGy and 50 kGy);

[0271] 2. Taq PCR Core Kit;

[0272] 3. ProofStart DNA polymerase;

[0273] 4. Prisms 3, 4, 9, 10, 15, 29, 30, 31, 36 and 37;

[0274] 5. Agarose;

[0275] 6. TAE Buffer;

[0276] 7. ethidium bromide.

[0277] Procedure: 1. Prepared PCR master mix as follows: 10x PCR buffer   5 μl dNTPs 1.39 μl  primers 1.39 μl  taq   1 μl ProofStart   1 μl water 33.22 μl  TSP 0.5 μl

[0278] 2. Aliquoted 43.61 μl of master mix into PCR tubes. Appropriate tubes contained the following primer pairs: (3, 4); (9, 10); (9, 15); (9, 29); (9, 30); (9, 31); (36, 37); and (9, 31), for both irradiated and unirradiated samples;

[0279] 3. Added 5 μl HBV per tube (irradiated or unirradiated);

[0280] 4. Ran PCR as follows:

[0281] 50° C. for 2 minutes (one cycle)

[0282] 95° C. for 2 minutes (one cycle)

[0283] 94° C. for 10 seconds (40 cycles)

[0284] 60° C. for 1 minute (40 cycles)

[0285] 68° C. for 2 minutes, five seconds (40 cycles);

[0286] 5. Ran 1% agarose gel (9 μl sample+1 μl sample buffer) at 100 v for about 20 minutes.

[0287] Results: Irradiated samples showed no band, indicating degradation of HBV clone by irradiation to 50 kGy.

Example 14

[0288] Purpose: To investigate the effect of gamma irradiation on samples containing HBV DNA and irradiated to 50 kGy.

[0289] Materials: 1. HBV DNA material (irradiated to 0 kGy and 50 kGy);

[0290] 2. Taq PCR Core Kit (Qiagen, cat. no. 201223);

[0291] 3. ProofStart Taq Polymerase (Qiagen, cat. no. 20);

[0292] 4. Prisms 10, 13, 30, 36 and 37 (FIG. 2);

[0293] 5. Agarose;

[0294] 6. TAE Buffer;

[0295] 7. Ethidium Bromide.

[0296] Procedure: 1. Prepared the following master mix: 10x buffer 60 μl dNTP 18 μl primer 36 16.68 μl   Taq 12 μl ProofStart 12 μl water 440.64 μl;  

[0297] 2. Pipetted 46.61 μl of master mix into PCR tubes;

[0298] 3. Added 1.39 μl of reverse primer (10, 13, 30 or 37) and 2 μl HBV DNA (0 kGy and 50 kGy) to appropriate tubes;

[0299] 4. Ran PCR for 50 cycles;

[0300] 5. Poured a 1% agarose gel (8 μl PCR product+1 μl sample buffer) at 100 V for about 20 minutes.

[0301] Results: Irradiated samples showed no band, indicating degradation of HBV DNA by irradiation to 50 kGy.

Example 15

[0302] Purpose: HBV amplification of nested primer set (about 80 bp, 400 bp and 697 bp) in samples containing ascorbate, including digestion of 0 kGy and 50 kGy samples with exonuclease I prior to PCR amplication.

[0303] Materials: 1. HBV DNA (irradiated to 0 kGy and 50 kGy, with and without ascorbate);

[0304] 2. Primer sets: (9, 10); (9, 14) and (9, 13) (FIG. 2);

[0305] 3. Exonuclease I;

[0306] 4. Standard PCR reagents.

[0307] Procedure: 1. Diluted HBV samples to 1/500, 1/2000 and 1/10000;

[0308] 2. Digested 1 μl raw HBV extract in 0.25 μl Exonuclease I, 10 μl 10× Exonuclease I buffer and 88.75 μl water at 37° C. for 30 minutes, inactivated at 80° C. for 20 minutes;

[0309] 3. Dilutes digested HBV to 1/2000 and 1/10000;

[0310] 4. Ran 55 cycles PCR.

[0311] Results: Irradiated and unirradiated samples coamplified with an 80 bp product. Only unirradiated samples amplified with a 697 bp product.

Example 16

[0312] Purpose: To investigate the amount of bacterial and fungal DNA present in pulverized tendon samples.

[0313] Materials: 1. E. Coli samples (tendon)−0 or 50 kGy+stabilizer (6.65×1¹⁰ CFU/μl);

[0314] 2. C. Albicans samples (tendon)−0 or 50 kGy+stabilizer (3.55×10⁹ CFU/μl);

[0315] 3. Staph. Aureus samples;

[0316] 4. Control tendon;

[0317] 5. Dneasey tissue kit (Qiagen, cat. no. 69504);

[0318] 6. Taq PCR Core Kit (Qiagen, cat. no. 201223);

[0319] 7. ProofStart Taq Polymerase (Qiagen, cat. no. 202205);

[0320] 8. Primers: Ribo 7, 8, 10, 11, 12, 13, 14 and Fungi 1, 2, 3, 4, 5, 6, 7, 8 (FIGS. 6A and 6B);

[0321] 9. Probes: FAM-RIBO Fungi Probe 5′-6FAM-TACAGTGAAACTGCGAATGGCTCATTAAATCAGTTA-TAMRA-3′;

[0322] 10. Microcon YM Centrifugal Filter Unit;

[0323] Procedure: 1. Using 0.05 tendon samples for E. coli and C. albicans, followed the Qiagen extraction profile;

[0324] 2. Prepared the following master mixes: Mix 1 Mix 2 10x buffer 150 μl  85 μl dNTPs 45 μl 25.5 μl   Ribo 741.7 — Fungi 1 — 23.65 μl   Taq 30 μl 17 μl ProofStart 30 μl 17 μl Water 936.6 μl   530.74 μl    FAM-RIBO 75 μl — Fungi Probe — 42.5 μl  

[0325] 3. Filtered master mixes using Microcon filter units for 30 minutes at 100×g;

[0326] 4. Pipetted 43.6 μl of Mix 1 into: rows A-D, columns 1-6; rows A-C, column 9; and row E, column 12;

[0327] 5. Pipetted 43.6 μl of Mix 2 into: rows E-F, columns 1-7 and row H, column 12;

[0328] 6. Pipetted 1.39 μl of reverse primer into appropriate well;

[0329] 7. Pipetted 5 μl DNA into appropriate wells;

[0330] 8. Ran PCR.

[0331] Results: Irradiation with 50 kGy resulted in decreased amplification of large target amplicons, indicating degradation of the pathogen genetic material caused by irradiation.

Example 17

[0332] Purpose: To show functionality of E. coli primers for RT-PCR using large target amphlicons.

[0333] Materials: 1. E. coli prepared from overnight culture;

[0334] 2. Dneasy Tissue Kit (Qiagen, cat. no. 96504); 3. Taq PCR Core Kit (Qiagen, cat. no. 201223)

[0335] 4. PtoofStart DNA polymerase (Qiagen, cat. no. 202205);

[0336] 5. Microcon YM-100 Centrifugal Filter Unit (cat. no. 42413);

[0337] 6. Primers: Ribo 1-9;

[0338] 7. Agarose;

[0339] 8. TAE Buffer;

[0340] 9. Ethidium Bromide.

[0341] Procedure: 1. Pipetted 1 ml of E. coli culture into each of 10 1.5 tubes;

[0342] 2. Centrifuged all 10 tubes for 5 minutes at maximum speed;

[0343] 3. Discarded supernatant;

[0344] 4. Placed 8 tubes in −80° C. and used 2 tubes for extraction following the Qiagen protocol;

[0345] 5. Prepared Master Mix as follows: 10x Buffer 5 μl dNTPs 1.5 μl pA 1.39 μl (Ribo 1 or 7) pB 1.39 μl (Ribo 2, 3, 4, 5, 6, 8 or 9) Taq 1 μl ProofStart 1 μl Water 33.22 μl TSP 0.5 μl

[0346] 6. Mixed Master Mix by inversion;

[0347] 7. Pipetted Master mix into a Microcon Centrifugal Filter Unit and centrifuged for 30 minutes at 100×g;

[0348] 8. Pipetted 43.61 μl of Master Mix into PCR tubes;

[0349] 9. Added appropriate reverse primer and DNA or water to create the following primer pairs: (1, 2)+5 μl DNA; (1, 2)+1 μl; (1, 3)+5 μl DNA; (1, 3)+1 μl DNA; (1, 4)+5 μl DNA; (1, 4)+1 μl DNA; (1, 5)+5 μl DNA; (1, 5)+1 μl DNA; (1, 6)+5 μL DNA; (1, 6)+1 μl DNA; (5, 8)+5 μl DNA; (7, 8)+1 μl DNA; (7, 9)+5 μl DNA; (7, 9)+1 μl DNA; and (1, 2)+5 (1, 4)+5 μl water;

[0350] 10. Ran PCR;

[0351] 11. Ran 1% Agarose gel at 100 V for about 20 min.

[0352] Results: All E. coli primers showed amplification of target sequences, regardless of size.

Example 18

[0353] Purpose: To investigate the effects of 50 kGy irradiation on samples containing E. coli.

[0354] Materials: 1. E. coli spiked tendon (irradiated to 0 kGy and 50 kGy)+6.65×10¹⁰ CFU/μl;

[0355] 2. Taq PCR Core Kit (Qiagen, cat. no. 201223);

[0356] 3. ProofStart Taq Polymerase (Qiagen, cat. no. 202205);

[0357] 4. Primers: Ribo 7, 8, 13, 14 and 15;

[0358] 5. Agarose;

[0359] 6. TAE Buffer;

[0360] 7. Ethidium Bromide;

[0361] 8. Microcon Centrifugal Filter Unit.

[0362] Procedure: 1. Prepared Master Mix as follows: 10x Buffer 60 μl dNTP 18 μl pA (forward) 16.68 μl   Taq 12 μl ProofStart 12 μl Water 452.64 μl;  

[0363] 2. Placed in Microcon and centrifuged for 30 minutes at 100×g;

[0364] 3. Pipetted 47-61 μl master mix into each or 9 PCR tubes;

[0365] 4. Added 1.39 μl of reverse primer and 1 μl DNA into appropriate tubes;

[0366] 5. Ran PCR.

[0367] 6. Ran 1% Agarose gel (8 μl sample+1 μl sample buffer) at 100 V for about 20 minutes.

[0368] Results: Samples irradiated to 50 kGy showed progessive disappearance of bands with increasing amplicon size, indicating degradation of the E. coli genetic material caused by irradiation.

Example 19

[0369] Purpose: To show functionality of Mt-DNA primers for RT-PCR using large target amplicons.

[0370] Materials: 1. Tendon DNA (irradiated to 0 kGy and 50 kGy);

[0371] 2. ROX 6 ({fraction (1/10)} dilution) molecular probes;

[0372] 3. Primers: MITO 1, 2, 3, 4, and 5 (FIG. 8);

[0373] 4. MITO Probe 1 (FIG. 8);

[0374] 5. Human DNA;

[0375] 6. Qiagen PCR Reagants;

[0376] 7. Qiagen ProofStart.

[0377] Procedure: 1. Prepared the following mixtures: Buffer 1.5 μl dNTPs 1.5 μl MITO 1 2.5 μl reverse primer 2.5 μl (MITO 2, 3, 4 or 5) MITO Probe 2.5 μl Taq   1 μl PS   1 μl 1/10 ROX   1 μl water  28 μl DNA   5 μl

[0378] 2. Ran 40 PCR;

[0379] 3. Ran 1% agatose gel (8 μl product+1 μl sample loading buffer) at 100 V for about one hour.

[0380] Results: Mt-DNA primers were functional, regardless of amplicon size.

[0381] Having now fully described this invention, it will be understood to those of ordinary skill in the art that the methods of the present invention can be carried out with a wide and equivalent range of conditions, formulations and other parameters without departing from the scope of the invention or any embodiments thereof. Moreover, the methods of the present invention may also be applied to situations other than the preferred embodiments described above. For example, instead of determining the level of potentially active pathogens, the methods described above may be used to determine the number of cells having an altered genetic sequence, such as turnout cells or genetically modified cells, in a tissue sample.

[0382] All patents and publications cited herein are hereby fully incorporated by reference in their entirety. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that such publication is prior art or that the present invention is not entitled to antedate such publication by virtue of prior invention.

1 99 1 5594 DNA B19 virus 1 ccaaatcaga tgccgccggt cgccgccggt aggcgggact tccggtacaa gatggcggac 60 aattacgtca tttcctgtga cgtcatttcc tgtgacgtca cttccggtgg gcgggacttc 120 cggaattagg gttggctctg ggccagcttg cttggggttg ccttgacact aagacaagcg 180 gcgcgccgct tgtcttagtg gcacgtcaac cccaagcgct ggcccagagc caaccctaat 240 tccggaagtc ccgcccaccg gaagtgacgt cacaggaaat gacgtcacag gaaatgacgt 300 aattgtccgc catcttgtac cggaagtccc gcctaccggc ggcgaccggc ggcatctgat 360 ttggtgtctt cttttaaatt ttagcgggct tttttcccgc cttatgcaaa tgggcagcca 420 ttttaagtgt ttcactataa ttttattggt cagttttgta acggttaaaa tgggcggagc 480 gtaggcgggg actacagtat atatagcacg gcactgccgc agctctttct ttctgggctg 540 ctttttcctg gactttcttg ctgttttttg tgagctaact aacaggtatt tatactactt 600 gttaacatac taacatggag ctatttagag gggtgcttca agtttcttct aatgttctgg 660 actgtgctaa cgataactgg tggtgctctt tactggattt agacacttct gactgggaac 720 cactaactca tactaacaga ctaatggcaa tatacttaag cagtgtggct tctaagcttg 780 actttaccgg ggggccacta gcggggtgct tgtacttttt tcaagtagaa tgtaacaaat 840 ttgaagaagg ctatcatatt catgtggtta ttggggggcc agggttaaac cccagaaacc 900 tcacagtgtg tgtagagggg ttatttaata atgtacttta tcaccttgta actgaaaatg 960 taaagctaaa atttttgcca ggaatgacta caaaaggcaa atactttaga gatggagagc 1020 agtttataga aaactattta atgaaaaaaa tacctttaaa tgttgtatgg tgtgttacta 1080 atattgatgg atatatagat acctgtattt ctgctacttt tagaagggga gcttgccatg 1140 ccaagaaacc ccgcattacc acagccataa atgacactag tagtgatgct ggggagtcta 1200 gcggcacagg ggcagaggtt gtgccaatta atgggaaggg aactaaggct agcataaagt 1260 ttcaaactat ggtaaactgg ttgtgtgaaa acagagtgtt tacagaggat aagtggaaac 1320 tagttgactt taaccagtac actttactaa gcagtagtca cagtggaagt tttcaaattc 1380 aaagtgcact aaaactagca atttataaag caactaattt agtgcctaca agcacatttc 1440 tattgcatac agactttgag caggttatgt gtattaaaga caataaaatt gttaaattgt 1500 tactttgtca aaactatgac cccctattag tggggcagca tgtgttaaag tggattgata 1560 aaaaatgtgg caagaaaaat acactgtggt tttatgggcc gccaagtaca ggaaaaacaa 1620 acttggcaat ggccattgct aaaagtgttc cagtatatgg catggttaac tggaataatg 1680 aaaactttcc atttaatgat gtagcaggga aaagcttggt ggtctgggat gaaggtatta 1740 ttaagtctac aattgtagaa gctgcaaaag ccattttagg cgggcaaccc accagggtag 1800 atcaaaaaat gcgtggaagt gtagctgtgc ctggagtacc tgtggttata accagcaatg 1860 gtgacattac ttttgttgta agcgggaaca ctacaacaac tgtacatgct aaagccttaa 1920 aagagcgaat ggtaaagtta aactttactg taagatgcag ccctgacatg gggttactaa 1980 cagaggctga tgtacaacag tggcttacat ggtgtaatgc acaaagctgg gaccactatg 2040 aaaactgggc aataaactac acttttgatt tccctggaat taatgcagat gccctccacc 2100 cagacctcca aaccacccca attgtcacag acaccagtat cagcagcagt ggtggtgaaa 2160 gctctgaaga actcagtgaa agcagctttt ttaacctcat caccccaggc gcctggaaca 2220 ctgaaacccc gcgctctagt acgcccatcc ccgggaccag ttcaggagaa tcatttgtcg 2280 gaagctcagt ttcctccgaa gttgtagctg catcgtggga agaagccttc tacacacctt 2340 tggcagacca gtttcgtgaa ctgttagttg gggttgatta tgtgtgggac ggtgtaaggg 2400 gtttacctgt gtgttgtgtg caacatatta acaatagtgg gggaggcttg ggactttgtc 2460 cccattgcat taatgtaggg gcttggtata atggatggaa atttcgagaa tttaccccag 2520 atttggtgcg gtgtagctgc catgtgggag cttctaatcc cttttctgtg ctaacctgca 2580 aaaaatgtgc ttacctgtct ggattgcaaa gctttgtaga ttatgagtaa agaaagtggc 2640 aaatggtggg aaagtgatga taaatttgct aaagctgtgt atcagcaatt tgtggaattt 2700 tatgaaaagg ttactggaac agacttagag cttattcaaa tattaaaaga tcactataat 2760 atttctttag ataatcccct agaaaaccca tcctctctgt ttgacttagt tgctcgtatt 2820 aaaaataacc ttaaaaactc tccagactta tatagtcatc attttcaaag tcatggacag 2880 ttatctgacc acccccatgc cttatcatcc agtagcagtc atgcagaacc tagaggagaa 2940 aatgcagtat tatctagtga agacttacac aagcctgggc aagttagcgt acaactaccc 3000 ggtactaact atgttgggcc tggcaatgag ctacaagctg ggcccccgca aagtgctgtt 3060 gacagtgctg caaggattca tgactttagg tatagccaac tggctaagtt gggaataaat 3120 ccatatactc attggactgt agcagatgaa gagcttttaa aaaatataaa aaatgaaact 3180 gggtttcaag cacaagtagt aaaagactac tttactttaa aaggtgcagc tgcccctgtg 3240 gcccattttc aaggaagttt gccggaagtt cccgcttaca acgcctcaga aaaataccca 3300 agcatgactt cagttaattc tgcagaagcc agcactggtg caggaggggg tggcagtaat 3360 cctgtcaaaa gcatgtggag tgagggggcc acttttagtg ccaactctgt aacttgtaca 3420 ttttccagac agtttttaat tccttatgac ccagagcacc attataaggt gttttctccc 3480 gcagcaagca gctgccacaa tgccagtgga aaggaggcaa aggtttgcac aattagtccc 3540 ataatgggat actcaacccc atggagatat ttagatttta atgctttaaa tttatttttt 3600 tcacctttag agtttcagca cttaattgaa aattatggaa gtatagctcc tgatgcttta 3660 actgtaacca tatcagaaat tgctgttaag gatgttacag acaaaactgg agggggggta 3720 caggttactg acagcactac agggcgccta tccatgttag tagaccatga atacaagtac 3780 ccatatgtgt taggacaagg tcaggatact ttagccccag aacttcctat ttgggtatac 3840 tttccccctc aatatgctta cttaacagta ggagatgtta acacacaagg aatctctgga 3900 gacagcaaaa aattagcaag tgaagaatca gcattttatg ttttggaaca cagttctttt 3960 cagcttttag gtacaggagg tacagcaact atgtcttata agtttcctcc agtgccccca 4020 gaaaatttag agggctgcag tcaacacttt tatgaaatgt acaatccctt atacggatcc 4080 cgcttagggg ttcctgacac attaggaggt gacccaaaat ttagatcttt aacacatgaa 4140 gaccatgcaa ttcagcccca aaacttcatg ccagggccac tagtaaactc agtgtctaca 4200 aaggagggag acagctctaa tactggagct ggaaaagcct taacaggcct tagcacaggc 4260 acctctcaaa acactagaat atccttacgc cctgggccag tgtcacagcc ataccaccac 4320 tgggacacag ataaatatgt tccaggaata aatgccattt ctcatggtca gaccacttat 4380 ggtaacgctg aagacaaaga gtatcagcaa ggagtgggta gatttccaaa tgaaaaagaa 4440 cagctaaaac agttacaggg tttaaacatg cacacctatt tccccaataa aggaacccag 4500 caatatacag atcaaattga gcgcccccta atggtgggtt ctgtatggaa cagaagagcc 4560 cttcactatg aaagccagct gtggagtaaa attccaaatt tagatgacag ttttaaaact 4620 cagtttgcag ccttaggagg atggggtttg catcagccac ctcctcaaat atttttaaaa 4680 atattaccac aaagtgggcc aattggaggt attaaatcaa tgggaattac taccttagtt 4740 cagtatgccg tgggaattat gacagtaact atgacattta aattggggcc ccgtaaagct 4800 acgggacggt ggaatcctca acctggagta tatcccccgc acgcagcagg tcatttacca 4860 tatgtactat atgaccccac agctacagat gcaaaacaac accacaggca tggatacgaa 4920 aagcctgaag aattgtggac agccaaaagc cgtgtgcacc cattgtaaac actccccacc 4980 gtgccctcag ccaggatgcg taactaaacg cccaccagta ccacccagac tgtacctgcc 5040 ccctcctgta cctataagac agcctaacac aaaagatata gacaatgtag aatttaagta 5100 cttaaccaga tatgaacaac atgttattag aatgttaaga ttgtgtaata tgtatcaaaa 5160 tttagaaaaa taaacatttg ttgtggttaa aaaattatgt tgttgcgctt taaaaattta 5220 aaagaagaca ccaaatcaga tgccgccggt cgccgccggt aggcgggact tccggtacaa 5280 gatggcggac aattacgtca tttcctgtga cgtcatttcc tgtgacgtca cttccggtgg 5340 gcgggacttc cggaattagg gttggctctg ggccagcgct tggggttgac gtgccactaa 5400 gacaagcggc gcgccgcttg tcttagtgtc aaggcaaccc caagcaagct ggcccagagc 5460 caaccctaat tccggaagtc ccgcccaccg gaagtgacgt cacaggaaat gacgtcacag 5520 gaaatgacgt aattgtccgc catcttgtac cggaagtccc gcctaccggc ggcgaccggc 5580 ggcatctgat ttgg 5594 2 3221 DNA Hepatitis B virus 2 ttccactgcc ttccaccaag ctctgcaaga ccccagagtc aggggtctgt attttcctgc 60 tggtggctcc agttcaggaa cagtaaaccc tgctccgaat attgcctctc acatctcgtc 120 aatctccgcg aggaccgggg accctgtgac gaacatggag aacatcacat caggattcct 180 aggacccctg cccgtgttac aggcggggtt tttcttgttg acaagaatcc tcacaatacc 240 gcagagtcta gactcgtggt ggacttctct caattttcta gggggatcac ccgtgtgtct 300 tggccaaaat tcgcgatccc caacctccaa tcactcacca acctcctgtc ctccaatttg 360 tcctggttat cgctggatgt gtctgcggcg ttttatcata ttcctcttca tcctgctgct 420 atgcctcatc ttcttattgg ttcttctgga ttatcaaggt atgttgcccg tttgtcctct 480 aattctagga tcaacaacaa ccagtacggg accatgcaaa acctgcacga ctcctgctca 540 aggcaactct atgtttccct catgttgctg tacaaaacct acggatggaa attgcacctg 600 tattcccatc ccatcgtctt gggctttcgc aaaataccta tgggagtggg cctcagtccg 660 tttctcttgg ctcagtttac tagtgccatt tgttcagtgg ttcgtagggc tttcccccac 720 tgtttggctt tcagctatat ggatgatgtg gtattggggg ccaagtctgt acagcatcgt 780 gagttccttt ataccgctgt taccaatttt cttttgtctc tgggtataca tttaaaccct 840 aacaaaacaa aaagatgggg ttattcccta aacttcatgg gttatgtaat tggaagttgg 900 ggaacattgc cacaggatca tattgtacaa aaaatcaaac actgttttag aaaacttcct 960 gttaacaggc ctattgattg gaaagtatgt caaagaattg tgggtctttt gggctttgct 1020 gctcctttta cacaatgtgg atatcctgcc ttaatgccct tgtatgcatg tatacaagct 1080 aaacaggctt tcactttctc gccaacttac aaggcctttc taagtaaaca gtacatgaac 1140 ctttaccccg ttgctcggca acggcctggt ctgtgccaag tatttgctga tgcaaccccc 1200 actggctggg gcttggccat aggccatcag cgcatgcgcg gaacctttgt ggctcctctg 1260 ccgatccata ctgcggaact cctagccgct tgttttgctc gcagccggtc tggagcgaaa 1320 ctcatcggaa ctgacaattc tgtcgtcctc tcgcggaaat atacctcgtt tccatggcta 1380 ctaggctgtg ctgccaactg gatccttcgc gggacgtcct ttgtttacgt cccgtcggcg 1440 ctgaatcccg cggacgaccc ctctcggggc cgcttgggac tctctcgtcc ccttctccgt 1500 ctgccgttcc agccgaccac ggggcgcacc tctctttacg cggtctcccc gtctgtgcct 1560 tctcatctgc cggtccgtgt gcacttcgct tcacctctgc acgttgcatg gagaccaccg 1620 tgaacgccca tcagatcctg cccaaggtct tacataagag gactcttgga ctcccagcaa 1680 tgtcaacgac cgaccttgag gcctacttca aagactgtgt gtttaaggac tgggaggagc 1740 tgggggagga gattaggtta aaggtctttg tattaggagg ctgtaggcat aaattggtct 1800 gcgcaccagc accatgcaac tttttcacct ctgcctaatc atctcttgta catgtcccac 1860 tgttcaagcc tccaagctgt gccttgggtg gctttggggc atggacattg acccttataa 1920 agaatttgga gctactgtgg agttactctc gtttttgcct tctgacttct ttccttccgt 1980 cagagatctc ctagacaccg cctcggctct gtatcgggaa gccttagagt ctcctgagca 2040 ttgctcacct caccataccg cactcaggca agccattctc tgctgggggg aattgatgac 2100 tctagctacc tgggtgggta ataatttgga agatccagca tccagggatc tagtagtcaa 2160 ttatgttaat actaacatgg gattaaagat caggcaactc ttgtggtttc atatctcttg 2220 ccttactttt ggaagagaaa ctgtacttga atatttggtc tctttcggag tgtggattcg 2280 cactcctcca gcctatagac caccaaatgc ccctatctta tcaacacttc cggaaactac 2340 tgttgttaga cgacgggacc gaggcaggtc ccctagaaga agaactccct cgcctcgcag 2400 acgcagatct caatcgccgc gtcgcagaag atctcaatct cgggaatctc aatgttagta 2460 ttccttggac tcataaggtg ggaaacttca ctgggcttta ttcctctaca gcacctatct 2520 ttaatcctga atggcaaact ccttcctttc ctaaaattca tttacaagag gacattatta 2580 ataggtgtca acaatttgtg ggccctctca ctgtaaatga aaagagaaga ttgaaattaa 2640 ttatgcctgc tagattctat cctacccaca ctaaatattt gcccttagac aaaggaatta 2700 aaccttatta tccagatcag gtagttaatc attacttcca aaccagacat tatttacata 2760 ctctttggaa ggcgggtatt ctatataaga gagaaaccac acgtagcgca tcattttgcg 2820 ggtcaccata ttcttgggaa caagagctac agcatgggag gttggtcatc aaaacctcgc 2880 aaaggcatgg ggacgaatct ttctgttccc aaccctctgg gattctttcc cgatcatcag 2940 ttggaccctg tattcggagc caactcaaac aatccagatt gggacttcaa ccccatcaag 3000 gaccactggc cagcagccaa ccaggtagga gtgggagcat tcgggccagg gttcacccct 3060 ccacacggcg gtgttttggg gtggagccct caggctcagg gcatgttgac cccagtgtca 3120 acaattcctc ctcctgcctc cgccaatcgg cagtcaggaa ggcagcctac tcccatctct 3180 ccacctctaa gagacagtca tcctcaggcc atgcagtgga a 3221 3 5075 DNA Porcine parvovirus 3 aatctttaaa ctgaccaact gtctttgcgt atggtgacgt gatgacgcgc gctacgcgcg 60 ctgccttcgg cagtcacacg tcaccatcag caaagacagt tggtcagttt aaagattaat 120 aagacattcc attggctgaa aagaggcggg aaattcaaaa aaagaggcgg gaaaaaaaga 180 ggtggagcct aacactataa atacagttgc ttacttcagt tagttccttt ctgcttcaga 240 ctgcacttcg ctccagagac acagctacaa actactctca gctactgcag catggcagcg 300 ggaaacactt actcggaaga ggtactaaaa gctaccaact ggcttcaaga taatgctcaa 360 aaagaagcat tctcttatgt atttaaaaca caaaaagtca atctaaatgg aaaagaaatt 420 gcttggaata actacaacaa agatacaaca gatgcggaaa tgataaacct acaaagagga 480 gcagaaacat catgggacca ggcaacagac atggaatggg aatcagaaat cgacagcctc 540 acaaaacggc aagtactgat ttttgactct cttgttaaaa aatgtctctt tgaaggtata 600 ttgcaaaaga acctaagtcc aagtgactgc tactggttca tacagcatga acatggtcaa 660 gatactggct atcactgcca tgtactacta ggtggaaaag gcttacaaca agcaatggga 720 aaatggttca gaaaacaatt aaacaattta tggagtagat ggttaataat gcaatgcaaa 780 gtacctctaa caccagttga aagaataaaa ttaagggaat tagcagagga tggtgagtgg 840 gtatcgctac taacctacac tcacaaacaa actaaaaaac aatatacaaa aatgactcat 900 tttggaaata tgattgctta ctacttccta aataaaaaaa gaaagacaac tgaaagagag 960 catggatatt atctcagctc agattctggc ttcatgacaa atttcttaaa agaaggcgag 1020 agacacttag tcagtcacct atttactgaa gcaaataaac ctgaaactgt ggaaacaacg 1080 gttactacag ctcaggaagc caaaagaggc agaatacaaa caaaaaaaga agtaagcata 1140 aaatgcacaa taagagactt ggttaataaa agatgtacta gcatagaaga ctggatgatg 1200 acagatccag acagttatat agaaatgatg gctcaaaccg gaggagaaaa tttaatcaaa 1260 aatacactag aaataacaac tcttactcta gcaagaacaa aaacagcata tgacttaata 1320 cttgaaaagg caaaaccaag catgctacca acatttaata ttagcaatac aagaacatgt 1380 aaaatattca gcatgcacaa ttggaactac attaaagtct gccatgctat aacttgtgta 1440 ctaaacagac aaggaggaaa aagaaataca attctatttc atgggccagc atcaacagga 1500 aaaagtataa ttgctcaaca cattgcaaac ttagttggta atgttggttg ctacaatgca 1560 gccaatgtga actttccatt taatgactgt acaaataaaa acttaatatg gattgaagaa 1620 gcaggaaact tctctaacca agtaaaccaa ttcaaagcca tatgttcagg tcaaacaatt 1680 agaattgacc aaaaaggtaa aggaagcaaa caaattgaac caactcctgt aataatgact 1740 acaaatgaag acataactaa agttagaata ggatgcgagg aaagaccaga acatacacaa 1800 ccaataagag acagaatgtt aaacataaac ctaaccagaa aactgccagg tgattttgga 1860 cttttagaag aaactgaatg gccactaata tgtgcttggt tggtaaagaa aggttaccaa 1920 gcaacaatgg ctagctatat gcatcattgg ggaaatgtac ctgattggtc agaaaaatgg 1980 gaggagccaa aaatgcaaac cccaataaat acaccaacag actctcagat ttccacatca 2040 gtgaaaactt cgccagcgga caacaactac gcagcaactc caatacagga ggacctggat 2100 ttagctttag ccttggagcc gtggagcgag ccaacaacac caactttcac caacctgcac 2160 ttaactccaa caccgccaga ttcagcaata cggacaccaa gtccaacttg gtcggaaata 2220 gaaaccgaca taagagcctg ctttggtgaa aactgtgcac ccacaacaaa ccttgaataa 2280 ggtaggatgg cgcctcctgc aaaaagagca agaggtaagg gtagttttaa gggggtggtg 2340 ggcatacata taaaactaac tgcaaataat ttttttatat attacaggac taactctacc 2400 aggatacaaa taccttggtc caggaaactc actagaccaa ggagaaccaa ctaatccatc 2460 agacgccgca gcaaaagaac acgacgaagc ctacgacaaa tacataaaat ctggaaaaaa 2520 tccatacttc tacttctcag cagctgatga aaaattcata aaagaaactg aacacgcaaa 2580 agactacgga ggtaaaattg gacattactt cttcagagca aagcgtgcct ttgctccaaa 2640 actctcagaa acagactcac caactacatc tcaacaacca gaggtaagaa gatcgccgag 2700 aaaacaccca gggtctaaac caccaggaaa aagacctgct ccaagacata tttttataaa 2760 cttagctaaa aaaaaagcta aagggacatc taatacaaac tctaactcaa tgagtgaaaa 2820 tgtggaacaa cacaacccta ttaatgcagg cactgaattg tctgcaacag gaaatgaatc 2880 tgggggtggg ggcggcggtg gcgggggtag gggtgctggg ggggttggtg tgtctacagg 2940 tactttcaat aatcaaacag aatttcaata cttgggggag ggcttggtta gaatcactgc 3000 acacgcatca agactcatac atctaaatat gccagaacac gaaacataca aaagaataca 3060 tgtactaaat tcagaatcag gggtggcggg acaaatggta caagacgatg cacacacaca 3120 aatggtaaca ccttggtcac taatagatgc taacgcatgg ggagtgtggt tcaatccagc 3180 ggactggcag ttaatatcca acaacatgac agaaataaac ttagttagtt ttgaacaaga 3240 aatattcaat gtagtactta aaacaattac agaatcagca acctcaccac caaccaaaat 3300 atataataat gatctaactg caagcttaat ggtcgcacta gacaccaata acacacttcc 3360 atacacacca gcagcaccta gaagtgaaac acttggtttt tatccatggt tacctacaaa 3420 accaactcaa tacagatatt acctatcatg catcagaaac ctaaatccac caacatacac 3480 tggacaatca caacaaataa cagactcaat acaaacagga ctacacagtg acattatgtt 3540 ctacacaata gaaaatgcag taccaattca tcttctaaga acaggagatg aattctccac 3600 aggaatatat cactttgaca caaaaccact aaaattaact cactcatggc aaacaaacag 3660 atctctagga ctgcctccaa aactactaac tgaacctacc acagaaggag accaacaccc 3720 aggaacacta ccagcagcta acacaagaaa aggttatcac caaacaatta ataatagcta 3780 cacagaagca acagcaatta ggccagctca ggtaggatat aatacaccat acatgaattt 3840 tgaatactcc aatggtggac catttctaac tcctatagta ccaacagcag acacacaata 3900 taatgatgat gaaccaaatg gtgctataag atttacaatg gattaccaac atggacactt 3960 aaccacatct tcacaagagc tagaaagata cacattcaat ccacaaagta aatgtggaag 4020 agctccaaag caacaattta atcaacaggc accactaaac ctagaaaata caaataatgg 4080 aacactttta ccttcagatc caataggagg gaaatctaac atgcatttca tgaatacact 4140 caatacatat ggaccattaa cagcactaaa caatactgca cctgtatttc caaatggtca 4200 aatatgggat aaagaacttg atacagatct aaaacctaga ctacatgtta cagctccatt 4260 tgtttgtaaa aacaatccac caggacaact atttgtaaaa atagcaccaa acctaacaga 4320 tgatttcaat gctgactctc ctcaacaacc tagaataata acttattcaa acttttggtg 4380 gaaaggaaca ctaacattca cagcaaaaat gagatccagt aatatgtgga accctattca 4440 acaacacaca acaacagcag aaaacattgg taactatatt cctacaaata ttggtggcat 4500 aagaatgttt ccagaatatt cacaacttat accaagaaaa ttatactaga aataactctg 4560 taaataaaaa ctcagttact tggttaatca tgtactacta tcattgtata cttcaataaa 4620 aataaattgt aaaatcaata aaactaagtt acttagtttc tgtataccta tactagaaat 4680 aactctgtaa ataaaaactc agttacttgg ttaatcatgt actactatca ttgtatactt 4740 caataaaaat aaattgtaaa atcaataaaa ctaagttact tagtttctgt ataccaatta 4800 tccccaaaaa acaataaaat tttaaaaaga aacaagctct catgtgttta ctattaacta 4860 aaccaaccac acttatatga ccttatgtct ttagggtggg tgggtgggaa ttactatgta 4920 ttcctttgag ttagttggtc gcctttgggc gactaaccaa gcggctctgc cgcttggtta 4980 gtcgcacggc gaccaactaa ctcaaaggaa tacatagtaa ttcccaccca cccaccctaa 5040 agacataagg tcatataagt gtggttggtt tagtt 5075 4 11703 DNA Sindbis virus 4 attgacggcg tagtacacac tattgaatca aacagccgac caattgcact accatcacaa 60 tggagaagcc agtagtaaac gtagacgtag acccccagag tccgtttgtc gtgcaactgc 120 aaaaaagctt cccgcaattt gaggtagtag cacagcaggt cactccaaat gaccatgcta 180 atgccagagc attttcgcat ctggccagta aactaatcga gctggaggtt cctaccacag 240 cgacgatctt ggacataggc agcgcaccgg ctcgtagaat gttttccgag caccagtatc 300 attgtgtctg ccccatgcgt agtccagaag acccggaccg catgatgaaa tacgccagta 360 aactggcgga aaaagcgtgc aagattacaa acaagaactt gcatgagaag attaaggatc 420 tccggaccgt acttgatacg ccggatgctg aaacaccatc gctctgcttt cacaacgatg 480 ttacctgcaa catgcgtgcc gaatattccg tcatgcagga cgtgtatatc aacgctcccg 540 gaactatcta tcatcaggct atgaaaggcg tgcggaccct gtactggatt ggcttcgaca 600 ccacccagtt catgttctcg gctatggcag gttcgtaccc tgcgtacaac accaactggg 660 ccgacgagaa agtccttgaa gcgcgtaaca tcggactttg cagcacaaag ctgagtgaag 720 gtaggacagg aaaattgtcg ataatgagga agaaggagtt gaagcccggg tcgcgggttt 780 atttctccgt aggatcgaca ctttatccag aacacagagc cagcttgcag agctggcatc 840 ttccatcggt gttccacttg aatggaaagc agtcgtacac ttgccgctgt gatacagtgg 900 tgagttgcga aggctacgta gtgaagaaaa tcaccatcag tcccgggatc acgggagaaa 960 ccgtgggata cgcggttaca cacaatagcg agggcttctt gctatgcaaa gttactgaca 1020 cagtaaaagg agaacgggta tcgttccctg tgtgcacgta catcccggcc accatatgcg 1080 atcagatgac tggtataatg gccacggata tatcacctga cgatgcacaa aaacttctgg 1140 ttgggctcaa ccagcgaatt gtcattaacg gtaggactaa caggaacacc aacaccatgc 1200 aaaattacct tctgccgatc atagcacaag ggttcagcaa atgggctaag gagcgcaagg 1260 atgatcttga taacgagaaa atgctgggta ctagagaacg caagcttacg tatggctgct 1320 tgtgggcgtt tcgcactaag aaagtacatt cgttttatcg cccacctgga acgcagacct 1380 gcgtaaaagt cccagcctct tttagcgctt ttcccatgtc gtccgtatgg acgacctctt 1440 tgcccatgtc gctgaggcag aaattgaaac tggcattgca accaaagaag gaggaaaaac 1500 tgctgcaggt ctcggaggaa ttagtcatgg aggccaaggc tgcttttgag gatgctcagg 1560 aggaagccag agcggagaag ctccgagaag cacttccacc attagtggca gacaaaggca 1620 tcgaggcagc cgcagaagtt gtctgcgaag tggaggggct ccaggcggac atcggagcag 1680 cattagttga aaccccgcgc ggtcacgtaa ggataatacc tcaagcaaat gaccgtatga 1740 tcggacagta tatcgttgtc tcgccaaact ctgtgctgaa gaatgccaaa ctcgcaccag 1800 cgcacccgct agcagatcag gttaagatca taacacactc cggaagatca ggaaggtacg 1860 cggtcgaacc atacgacgct aaagtactga tgccagcagg aggtgccgta ccatggccag 1920 aattcctagc actgagtgag agcgccacgt tagtgtacaa cgaaagagag tttgtgaacc 1980 gcaaactata ccacattgcc atgcatggcc ccgccaagaa tacagaagag gagcagtaca 2040 aggttacaaa ggcagagctt gcagaaacag agtacgtgtt tgacgtggac aagaagcgtt 2100 gcgttaagaa ggaagaagcc tcaggtctgg tcctctcggg agaactgacc aaccctccct 2160 atcatgagct agctctggag ggactgaaga cccgacctgc ggtcccgtac aaggtcgaaa 2220 caataggagt gataggcaca ccggggtcgg gcaagtcagc tattatcaag tcaactgtca 2280 cggcacgaga tcttgttacc agcggaaaga aagaaaattg tcgcgaaatt gaggccgacg 2340 tgctaagact gaggggtatg cagattacgt cgaagacagt agattcggtt atgctcaacg 2400 gatgccacaa agccgtagaa gtgctgtacg ttgacgaagc gttcgcgtgc cacgcaggag 2460 cactacttgc cttgattgct atcgtcaggc cccgcaagaa ggtagtacta tgcggagacc 2520 ccatgcaatg cggattcttc aacatgatgc aactaaaggt acatttcaat caccctgaaa 2580 aagacatatg caccaagaca ttctacaagt atatctcccg gcgttgcaca cagccagtta 2640 cagctattgt atcgacactg cattacgatg gaaagatgaa aaccacgaac ccgtgcaaga 2700 agaacattga aatcgatatt acaggggcca caaagccgaa gccaggggat atcatcctga 2760 catgtttccg cgggtgggtt aagcaattgc aaatcgacta tcccggacat gaagtaatga 2820 cagccgcggc ctcacaaggg ctaaccagaa aaggagtgta tgccgtccgg caaaaagtca 2880 atgaaaaccc actgtacgcg atcacatcag agcatgtgaa cgtgttgctc acccgcactg 2940 aggacaggct agtgtggaaa accttgcagg gcgacccatg gattaagcag cccactaaca 3000 tacctaaagg aaactttcag gctactatag aggactggga agctgaacac aagggaataa 3060 ttgctgcaat aaacagcccc actccccgtg ccaatccgtt cagctgcaag accaacgttt 3120 gctgggcgaa agcattggaa ccgatactag ccacggccgg tatcgtactt accggttgcc 3180 agtggagcga actgttccca cagtttgcgg atgacaaacc acattcggcc atttacgcct 3240 tagacgtaat ttgcattaag tttttcggca tggacttgac aagcggactg ttttctaaac 3300 agagcatccc actaacgtac catcccgccg attcagcgag gccggtagct cattgggaca 3360 acagcccagg aacccgcaag tatgggtacg atcacgccat tgccgccgaa ctctcccgta 3420 gatttccggt gttccagcta gctgggaagg gcacacaact tgatttgcag acggggagaa 3480 ccagagttat ctctgcacag cataacctgg tcccggtgaa ccgcaatctt cctcacgcct 3540 tagtccccga gtacaaggag aagcaacccg gcccggtcaa aaaattcttg aaccagttca 3600 aacaccactc agtacttgtg gtatcagagg aaaaaattga agctccccgt aagagaatcg 3660 aatggatcgc cccgattggc atagccggtg cagataagaa ctacaacctg gctttcgggt 3720 ttccgccgca ggcacggtac gacctggtgt tcatcaacat tggaactaaa tacagaaacc 3780 accactttca gcagtgcgaa gaccatgcgg cgaccttaaa aaccctttcg cgttcggccc 3840 tgaattgcct taacccagga ggcaccctcg tggtgaagtc ctatggctac gccgaccgca 3900 acagtgagga cgtagtcacc gctcttgcca gaaagtttgt cagggtgtct gcagcgagac 3960 cagattgtgt ctcaagcaat acagaaatgt acctgatttt ccgacaacta gacaacagcc 4020 gtacacggca attcaccccg caccatctga attgcgtgat ttcgtccgtg tatgagggta 4080 caagagatgg agttggagcc gcgccgtcat accgcaccaa aagggagaat attgctgact 4140 gtcaagagga agcagttgtc aacgcagcca atccgctggg tagaccaggc gaaggagtct 4200 gccgtgccat ctataaacgt tggccgacca gttttaccga ttcagccacg gagacaggca 4260 ccgcaagaat gactgtgtgc ctaggaaaga aagtgatcca cgcggtcggc cctgatttcc 4320 ggaagcaccc agaagcagaa gccttgaaat tgctacaaaa cgcctaccat gcagtggcag 4380 acttagtaaa tgaacataac atcaagtctg tcgccattcc actgctatct acaggcattt 4440 acgcagccgg aaaagaccgc cttgaagtat cacttaactg cttgacaacc gcgctagaca 4500 gaactgacgc ggacgtaacc atctattgcc tggataagaa gtggaaggaa agaatcgacg 4560 cggcactcca acttaaggag tctgtaacag agctgaagga tgaagatatg gagatcgacg 4620 atgagttagt atggatccat ccagacagtt gcttgaaggg aagaaaggga ttcagtacta 4680 caaaaggaaa attgtattcg tacttcgaag gcaccaaatt ccatcaagca gcaaaagaca 4740 tggcggagat aaaggtcctg ttccctaatg accaggaaag taatgaacaa ctgtgtgcct 4800 acatattggg tgagaccatg gaagcaatcc gcgaaaagtg cccggtcgac cataacccgt 4860 cgtctagccc gcccaaaacg ttgccgtgcc tttgcatgta tgccatgacg ccagaaaggg 4920 tccacagact tagaagcaat aacgtcaaag aagttacagt atgctcctcc accccccttc 4980 ctaagcacaa aattaagaat gttcagaagg ttcagtgcac gaaagtagtc ctgtttaatc 5040 cgcacactcc cgcattcgtt cccgcccgta agtacataga agtgccagaa cagcctaccg 5100 ctcctcctgc acaggccgag gaggcccccg aagttgtagc gacaccgtca ccatctacag 5160 ctgataacac ctcgcttgat gtcacagaca tctcactgga tatggatgac agtagcgaag 5220 gctcactttt ttcgagcttt agcggatcgg acaactctat tactagtatg gacagttggt 5280 cgtcaggacc tagttcacta gagatagtag accgaaggca ggtggtggtg gctgacgttc 5340 atgccgtcca agagcctgcc cctattccac cgccaaggct aaagaagatg gcccgcctgg 5400 cagcggcaag aaaagagccc actccaccgg caagcaatag ctctgagtcc ctccacctct 5460 cttttggtgg ggtatccatg tccctcggat caattttcga cggagagacg gcccgccagg 5520 cagcggtaca acccctggca acaggcccca cggatgtgcc tatgtctttc ggatcgtttt 5580 ccgacggaga gattgatgag ctgagccgca gagtaactga gtccgaaccc gtcctgtttg 5640 gatcatttga accgggcgaa gtgaactcaa ttatatcgtc ccgatcagcc gtatcttttc 5700 cactacgcaa gcagagacgt agacgcagga gcaggaggac tgaatactga ctaaccgggg 5760 taggtgggta catattttcg acggacacag gccctgggca cttgcaaaag aagtccgttc 5820 tgcagaacca gcttacagaa ccgaccttgg agcgcaatgt cctggaaaga attcatgccc 5880 cggtgctcga cacgtcgaaa gaggaacaac tcaaactcag gtaccagatg atgcccaccg 5940 aagccaacaa aagtaggtac cagtctcgta aagtagaaaa tcagaaagcc ataaccactg 6000 agcgactact gtcaggacta cgactgtata actctgccac agatcagcca gaatgctata 6060 agatcaccta tccgaaacca ttgtactcca gtagcgtacc ggcgaactac tccgatccac 6120 agttcgctgt agctgtctgt aacaactatc tgcatgagaa ctatccgaca gtagcatctt 6180 atcagattac tgacgagtac gatgcttact tggatatggt agacgggaca gtcgcctgcc 6240 tggatactgc aaccttctgc cccgctaagc ttagaagtta cccgaaaaaa catgagtata 6300 gagccccgaa tatccgcagt gcggttccat cagcgatgca gaacacgcta caaaatgtgc 6360 tcattgccgc aactaaaaga aattgcaacg tcacgcagat gcgtgaactg ccaacactgg 6420 actcagcgac attcaatgtc gaatgctttc gaaaatatgc atgtaatgac gagtattggg 6480 aggagttcgc tcggaagcca attaggatta ccactgagtt tgtcaccgca tatgtagcta 6540 gactgaaagg ccctaaggcc gccgcactat ttgcaaagac gtataatttg gtcccattgc 6600 aagaagtgcc tatggataga ttcgtcatgg acatgaaaag agacgtgaaa gttacaccag 6660 gcacgaaaca cacagaagaa agaccgaaag tacaagtgat acaagccgca gaacccctgg 6720 cgactgctta cttatgcggg attcaccggg aattagtgcg taggcttacg gccgtcttgc 6780 ttccaaacat tcacacgctt tttgacatgt cggcggagga ttttgatgca atcatagcag 6840 aacacttcaa gcaaggcgac ccggtactgg agacggatat cgcatcattc gacaaaagcc 6900 aagacgacgc tatggcgtta accggtctga tgatcttgga ggacctgggt gtggatcaac 6960 cactactcga cttgatcgag tgcgcctttg gagaaatatc atccacccat ctacctacgg 7020 gtactcgttt taaattcggg gcgatgatga aatccggaat gttcctcaca ctttttgtca 7080 acacagtttt gaatgtcgtt atcgccagca gagtactaga agagcggctt aaaacgtcca 7140 gatgtgcagc gttcattggc gacgacaaca tcatacatgg agtagtatct gacaaagaaa 7200 tggctgagag gtgcgccacc tggctcaaca tggaggttaa gatcatcgac gcagtcatcg 7260 gtgagagacc accttacttc tgcggcggat ttatcttgca agattcggtt acttccacag 7320 cgtgccgcgt ggcggatccc ctgaaaaggc tgtttaagtt gggtaaaccg ctcccagccg 7380 acgacgagca agacgaagac agaagacgcg ctctgctaga tgaaacaaag gcgtggttta 7440 gagtaggtat aacaggcact ttagcagtgg ccgtgacgac ccggtatgag gtagacaata 7500 ttacacctgt cctactggca ttgagaactt ttgcccagag caaaagagca ttccaagcca 7560 tcagagggga aataaagcat ctctacggtg gtcctaaata gtcagcatag tacatttcat 7620 ctgactaata ctacaacacc accaccatga atagaggatt ctttaacatg ctcggccgcc 7680 gccccttccc ggcccccact gccatgtgga ggccgcggag aaggaggcag gcggccccga 7740 tgcctgcccg caacgggctg gcttctcaaa tccagcaact gaccacagcc gtcagtgccc 7800 tagtcattgg acaggcaact agacctcaac ccccacgtcc acgcccgcca ccgcgccaga 7860 agaagcaggc gcccaagcaa ccaccgaagc cgaagaaacc aaaaacgcag gagaagaaga 7920 agaagcaacc tgcaaaaccc aaacccggaa agagacagcg catggcactt aagttggagg 7980 ccgacagatt gttcgacgtc aagaacgagg acggagatgt catcgggcac gcactggcca 8040 tggaaggaaa ggtaatgaaa cctctgcacg tgaaaggaac catcgaccac cctgtgctat 8100 caaagctcaa atttaccaag tcgtcagcat acgacatgga gttcgcacag ttgccagtca 8160 acatgagaag tgaggcattc acctacacca gtgaacaccc cgaaggattc tataactggc 8220 accacggagc ggtgcagtat agtggaggta gatttaccat ccctcgcgga gtaggaggca 8280 gaggagacag cggtcgtccg atcatggata actccggtcg ggttgtcgcg atagtcctcg 8340 gtggcgctga tgaaggaaca cgaactgccc tttcggtcgt cacctggaat agtaaaggga 8400 agacaattaa gacgaccccg gaagggacag aagagtggtc cgcagcacca ctggtcacgg 8460 caatgtgttt gctcggaaat gtgagcttcc catgcgaccg cccgcccaca tgctataccc 8520 gcgaaccttc cagagccctc gacatccttg aagagaacgt gaaccatgag gcctacgata 8580 ccctgctcaa tgccatattg cggtgcggat cgtctggcag aagcaaaaga agcgtcattg 8640 acgactttac cctgaccagc ccctacttgg gcacatgctc gtactgccac catactgtac 8700 cgtgcttcag ccctgttaag atcgagcagg tctgggacga agcggacgat aacaccatac 8760 gcatacagac ttccgcccag tttggatacg accaaagcgg agcagcaagc gcaaacaagt 8820 accgctacat gtcgcttaag caggatcaca ccgttaaaga aggcaccatg gatgacatca 8880 agattagcac ctcaggaccg tgtagaaggc ttagctacaa aggatacttt ctcctcgcaa 8940 aatgccctcc aggggacagc gtaacggtta gcatagtgag tagcaactca gcaacgtcat 9000 gtacactggc ccgcaagata aaaccaaaat tcgtgggacg ggaaaaatat gatctacctc 9060 ccgttcacgg taaaaaaatt ccttgcacag tgtacgaccg tctgaaagaa acaactgcag 9120 gctacatcac tatgcacagg ccgagaccgc acgcttatac atcctacctg gaagaatcat 9180 cagggaaagt ttacgcaaag ccgccatctg ggaagaacat tacgtatgag tgcaagtgcg 9240 gcgactacaa gaccggaacc gtttcgaccc gcaccgaaat cactggttgc accgccatca 9300 agcagtgcgt cgcctataag agcgaccaaa cgaagtgggt cttcaactca ccggacttga 9360 tcagacatga cgaccacacg gcccaaggga aattgcattt gcctttcaag ttgatcccga 9420 gtacctgcat ggtccctgtt gcccacgcgc cgaatgtaat acatggcttt aaacacatca 9480 gcctccaatt agatacagac cacttgacat tgctcaccac caggagacta ggggcaaacc 9540 cggaaccaac cactgaatgg atcgtcggaa agacggtcag aaacttcacc gtcgaccgag 9600 atggcctgga atacatatgg ggaaatcatg agccagtgag ggtctatgcc caagagtcag 9660 caccaggaga ccctcacgga tggccacacg aaatagtaca gcattactac catcgccatc 9720 ctgtgtacac catcttagcc gtcgcatcag ctaccgtggc gatgatgatt ggcgtaactg 9780 ttgcagtgtt atgtgcctgt aaagcgcgcc gtgagtgcct gacgccatac gccctggccc 9840 caaacgccgt aatcccaact tcgctggcac tcttgtgctg cgttaggtcg gccaatgctg 9900 aaacgttcac cgagaccatg agttacttgt ggtcgaacag tcagccgttc ttctgggtcc 9960 agttgtgcat acctttggcc gctttcatcg ttctaatgcg ctgctgctcc tgctgcctgc 10020 cttttttagt ggttgccggc gcctacctgg cgaaggtaga cgcctacgaa catgcgacca 10080 ctgttccaaa tgtgccacag ataccgtata aggcacttgt tgaaagggca gggtatgccc 10140 cgctcaattt ggagatcact gtcatgtcct cggaggtttt gccttccacc aaccaagagt 10200 acattacctg caaattcacc actgtggtcc cctccccaaa aatcaaatgc tgcggctcct 10260 tggaatgtca gccggccgct catgcagact atacctgcaa ggtcttcgga ggggtctacc 10320 cctttatgtg gggaggagcg caatgttttt gcgacagtga gaacagccag atgagtgagg 10380 cgtacgtcga attgtcagca gattgcgcgt ctgaccacgc gcaggcgatt aaggtgcaca 10440 ctgccgcgat gaaagtagga ctgcgtattg tgtacgggaa cactaccagt ttcctagatg 10500 tgtacgtgaa cggagtcaca ccaggaacgt ctaaagactt gaaagtcata gctggaccaa 10560 tttcagcatc gtttacgcca ttcgatcata aggtcgttat ccatcgcggc ctggtgtaca 10620 actatgactt cccggaatat ggagcgatga aaccaggagc gtttggagac attcaagcta 10680 cctccttgac tagcaaggat ctcatcgcca gcacagacat taggctactc aagccttccg 10740 ccaagaacgt gcatgtcccg tacacgcagg cctcatcagg atttgagatg tggaaaaaca 10800 actcaggccg cccactgcag gaaaccgcac ctttcgggtg taagattgca gtaaatccgc 10860 tccgagcggt ggactgttca tacgggaaca ttcccatttc tattgacatc ccgaacgctg 10920 cctttatcag gacatcagat gcaccactgg tctcaacagt caaatgtgaa gtcagtgagt 10980 gcacttattc agcagacttc ggcgggatgg ccaccctgca gtatgtatcc gaccgcgaag 11040 gtcaatgccc cgtacattcg cattcgagca cagcaactct ccaagagtcg acagtacatg 11100 tcctggagaa aggagcggtg acagtacact ttagcaccgc gagtccacag gcgaacttta 11160 tcgtatcgct gtgtgggaag aagacaacat gcaatgcaga atgtaaacca ccagctgacc 11220 atatcgtgag caccccgcac aaaaatgacc aagaatttca agccgccatc tcaaaaacat 11280 catggagttg gctgtttgcc cttttcggcg gcgcctcgtc gctattaatt ataggactta 11340 tgatttttgc ttgcagcatg atgctgacta gcacacgaag atgaccgcta cgccccaatg 11400 atccgaccag caaaactcga tgtacttccg aggaactgat gtgcataatg catcaggctg 11460 gtacattaga tccccgctta ccgcgggcaa tatagcaaca ctaaaaactc gatgtacttc 11520 cgaggaagcg cagtgcataa tgctgcgcag tgttgccaca taaccactat attaaccatt 11580 tatctagcgg acgccaaaaa ctcaatgtat ttctgaggaa gcgtggtgca taatgccacg 11640 cagcgtctgc ataactttta ttatttcttt tattaatcaa caaaattttg tttttaacat 11700 ttc 11703 5 10945 DNA West Nile virus 5 gaggattaac aacaattaac acagtgcgag ctgtttctta gcacgaagat ctcgatgtct 60 aagaaaccag gagggcccgg caagagccgg gctgtcaata tgctaaaacg cggaatgccc 120 cgcgtgttgt ccttgattgg actgaagagg gctatgttga gcctgatcga cggcaagggg 180 ccaatacgat ttgtgttggc tctcttggcg ttcttcaggt tcacagcaat tgctccgacc 240 cgagcagtgc tggatcgatg gagaggtgtg aacaaacaaa cagcgatgaa acaccttctg 300 agttttaaga aggaactagg gaccttgacc agtgctatca atcggcggag ctcaaaacaa 360 aagaaaagag gaggaaagac cggaattgca gtcatgattg gcctgatcgc cagcgtagga 420 gcagttaccc tctctaactt ccaagggaag gtgatgatga cggtaaatgc tactgacgtc 480 acagatgtca tcacgattcc aacagctgct ggaaagaacc tatgcattgt cagagcaatg 540 gatgtgggat acatgtgcga tgatactatc acctatgaat gcccagtgct gtcggctggt 600 aatgatccag aagacatcga ctgttggtgc acaaagtcag cagtctacgt caggtatgga 660 agatgcacca agacacgcca ctcaagacgc agtcggaggt cactgacagt gcagacacac 720 ggagaaagca ctctagcgaa caagaagggg gcttggatgg acagcaccaa ggccacaagg 780 tatttggtaa aaacagaatc atggatcttg aggaaccctg gatatgccct ggtggcagcc 840 gtcattggtt ggatgcttgg gagcaacacc atgcagagag ttgtgtttgt cgtgctattg 900 cttttggtgg ccccagctta cagcttcaac tgccttggaa tgagcaacag agacttcttg 960 gaaggagtgt ctggagcaac atgggtggat ttggttctcg aaggcgatag ctgcgtgact 1020 atcatgtcta aggacaagcc taccatcgat gtgaagatga tgaatatgga ggcggccaac 1080 ctggcagagg tccgcagtta ttgctatttg gctaccgtca gcgatctctc caccaaagct 1140 gcgtgcccga ccatggggga agcccacaat gacaaacgtg ctgacccagc ttttgtgtgc 1200 agacaaggag tggtggacag gggctggggc aacggctgcg gactatttgg caaaggaagc 1260 attgacacat gcgccaaatt tgcctgctct accaaggcaa taggaagaac catcttgaaa 1320 gagaatatca agtacgaagt ggccattttt gtccatggac caactactgt ggagtcgcac 1380 ggaaactact ccacacaggt tggagccact caggcaggga gattcagcat cactcctgcg 1440 gcgccttcat acacactaaa gcttggagaa tatggagagg tgacagtgga ctgtgaacca 1500 cggtcaggga ttgacaccaa tgcatactac gtgatgactg ttggaacaaa gacgttcttg 1560 gtccatcgtg agtggttcat ggacctcaac ctcccttgga gcagtgctgg aagtactgtg 1620 tggaggaaca gagagacgtt aatggagttt gaggaaccac acgccacgaa gcagtctgtg 1680 atagcattgg gctcacaaga gggagctctg catcaagctt tggctggagc cattcctgtg 1740 gaattttcaa gcaacactgt caagttgacg tcgggtcatt tgaagtgtag agtgaagatg 1800 gaaaaattgc agttgaaggg aacaacctat ggcgtctgtt caaaggcttt caagtttctt 1860 gggactcccg cagacacagg tcacggcact gtggtgttgg aattgcagta cactggcacg 1920 gatggacctt gcaaagttcc tatctcgtca gtggcttcat tgaacgacct aacgccagtg 1980 ggcagattgg tcactgtcaa cccttttgtt tcagtggcca cggccaacgc taaggtcctg 2040 attgaattgg aaccaccctt tggagactca tacatagtgg tgggcagagg agaacaacag 2100 atcaatcacc attggcacaa gtctggaagc agcattggca aagcctttac aaccaccctc 2160 aaaggagcgc agagactagc cgctctagga gacacagctt gggactttgg atcagttgga 2220 ggggtgttca cctcagttgg gaaggctgtc catcaagtgt tcggaggagc attccgctca 2280 ctgttcggag gcatgtcctg gataacgcaa ggattgctgg gggctctcct gttgtggatg 2340 ggcatcaatg ctcgtgatag gtccatagct ctcacgtttc tcgcagttgg aggagttctg 2400 ctcttcctct ccgtgaacgt gcacgctgac actgggtgtg ccataaacat cacccggcaa 2460 gagctgagat gtggaagtgg agtgttcata cacaatgatg tggaggcttg gatggaccgg 2520 tacaagtatt accctgaaac gccacaaggc ctagccaaga tcattcagaa agctcataag 2580 gaaggagtgt gcggtctacg atcagtttcc agactggagc atcaaatgtg ggaagcagtg 2640 aaggacgagc tgaacactcc tttgaaggag aatggtgtgg accttagtgt cgtggttgag 2700 aaacaggagg gaatgtacaa gtcagcacct aaacgcctca ccgccaccac ggaaaaattg 2760 gaaattggct ggaaggcctg gggaaagagt attttatttg caccagaact cgccaacaac 2820 acctttgtgg ttgatggtcc ggagaccaag gaatgtccga ctcagaatcg cgcttggaat 2880 agcttagaag tggaggattt tggatttggt ctcaccagca ctcggatgtt cctgaaggtc 2940 agagaaggca acacaactga atgtgactcg aagatcattg gaacggctgt caagaacaac 3000 ttggcgatcc acagtgacct gtcctattgg attgaaagca ggctcaatga tacgtggaag 3060 cttgaaaggg cagttctggg tgaagtcaaa tcatgtacgt ggcctgagac gcataccttg 3120 tggggcgatg gaatccttga gagtgacttg ataataccag tcacactggc gggaccacga 3180 agcaatcaca atcggagacc tgggtacaag acacaaaacc agggcccatg ggacgaaggc 3240 cgggtagaga ttgacttcga ttactgccca ggaactacgg tcaccctgag tgagagctgc 3300 ggacaccgtg gacctgccac tcgcaccacc acagagagcg gaaagttgat aacagattgg 3360 tgctgcagga gctgcacctt accaccactg cgctaccaaa ctgacagcgg ctgttggtat 3420 ggtatggaga tcagaccaca gagacatgat gaaaagaccc tcgtgcagtc acaagtgaat 3480 gcttataatg ctgatatgat tgaccctttt cagttgggcc ttctggtcgt gttcttggcc 3540 acccaggagg tccttcgcaa gaggtggaca gccaagatca gcatgccagc tatactgatt 3600 gctctgctag tcctggtgtt tgggggcatt acttacactg atgtgttacg ctatgtcatc 3660 ttggtggggg cagctttcgc agaatctaat tcgggaggag acgtggtaca cttggcgctc 3720 atggcgacct tcaagataca accagtgttt atggtggcat cgtttctcaa agcgagatgg 3780 accaaccagg agaacatttt gttgatgttg gcggctgttt tctttcaaat ggcttatcac 3840 gatgcccgcc aaattctgct ctgggagatc cctgatgtgt tgaattcact ggcggtagct 3900 tggatgatac tgagagccat aacattcaca acgacatcaa acgtggttgt tccgctgcta 3960 gccctgctaa cacccgggct gagatgcttg aatctggatg tgtacaggat actgctgttg 4020 atggtcggaa taggcagctt gatcagggag aagaggagtg cagctgcaaa aaagaaagga 4080 gcaagtctgc tatgcttggc tctagcctca acaggacttt tcaaccccat gatccttgct 4140 gctggactga ttgcatgtga tcccaaccgt aaacgcggat ggcccgcaac tgaagtgatg 4200 acagctgtcg gcctaatgtt tgccatcgtc ggagggctgg cagagcttga cattgactcc 4260 atggccattc caatgactat cgcggggctc atgtttgctg ctttcgtgat ttctgggaaa 4320 tcaacagata tgtggattga gagaacggcg gacatttcct gggaaagtga tgcagaaatt 4380 acaggctcga gcgaaagagt tgatgtgcgg cttgatgatg atggaaactt ccagctcatg 4440 aatgatccag gagcaccttg gaagatatgg atgctcagaa tggtctgtct cgcgattagt 4500 gcgtacaccc cctgggcaat cttgccctca gtagttggat tttggataac tctccaatac 4560 acaaagagag gaggcgtgtt gtgggacact ccctcaccaa aggagtacaa aaagggggac 4620 accaccaccg gcgtttacag gatcatgact cgtgggctgc tcggcagtta tcaagcagga 4680 gcgggcgtga tggttgaagg tgttttccac accctttggc atacaacaaa aggagccgct 4740 ttgatgagcg gagagggccg cctggaccca tactggggca gtgtcaagga ggatcgactt 4800 tgttacggag gaccctggaa attgcagcac aagtggaacg ggcaggatga ggtgcagatg 4860 attgtggtgg aacctggcaa gaacgttaag aacgtccaga cgaaaccagg ggtgttcaaa 4920 acacctgaag gagaaatcgg ggccgtgact ttggacttcc ccactggaac atcaggctca 4980 ccaatagtgg acaaaaacgg tgatgtgatt gggctttatg gcaatggagt cataatgccc 5040 aacggctcat acataagcgc gatagtgcag ggtgaaagga tggatgagcc aatcccagcc 5100 ggattcgaac ctgagatgct gaggaaaaaa cagatcactg tactggatct ccatcccggc 5160 gccggtaaaa caaggaggat tctgccacag atcatcaaag aggccataaa cagaagactg 5220 agaacagccg tgctagcacc aaccagggtt gtggctgctg agatggctga agcactgaga 5280 ggactgccca tccggtacca gacatccgca gtgcccagag aacataatgg aaatgagatt 5340 gttgatgtca tgtgtcatgc taccctcacc cacaggctga tgtctcctca cagggtgccg 5400 aactacaacc tgttcgtgat ggatgaggct catttcaccg acccagctag cattgcagca 5460 agaggttaca tttccacaaa ggtcgagcta ggggaggcgg cggcaatatt catgacagcc 5520 accccaccag gcacttcaga tccattccca gagtccaatt caccaatttc cgacttacag 5580 actgagatcc cggatcgagc ttggaactct ggatacgaat ggatcacaga atacaccggg 5640 aagacggttt ggtttgtgcc tagtgtcaag atggggaatg agattgccct ttgcctacaa 5700 cgtgctggaa agaaagtagt ccaattgaac agaaagtcgt acgagacgga gtacccaaaa 5760 tgtaagaacg atgattggga ctttgttatc acaacagaca tatctgaaat gggggctaac 5820 ttcaaggcga gcagggtgat tgacagccgg aagagtgtga aaccaaccat cataacagaa 5880 ggagaaggga gagtgatcct gggagaacca tctgcagtga cagcagctag tgccgcccag 5940 agacgtggac gtatcggtag aaatccgtcg caagttggtg atgagtactg ttatgggggg 6000 cacacgaatg aagacgactc gaacttcgcc cattggactg aggcacgaat catgctggac 6060 aacatcaaca tgccaaacgg actgatcgct caattctacc aaccagagcg tgagaaggta 6120 tataccatgg atggggaata ccggctcaga ggagaagaga gaaaaaactt tctggaactg 6180 ttgaggactg cagatctgcc agtttggctg gcttacaagg ttgcagcggc tggagtgtca 6240 taccacgacc ggaggtggtg ctttgatggt cctaggacaa acacaatttt agaagacaac 6300 aacgaagtgg aagtcatcac gaagcttggt gaaaggaaga ttctgaggcc gcgctggatt 6360 gacgccaggg tgtactcgga tcaccaggca ctaaaggcgt tcaaggactt cgcctcggga 6420 aaacgttctc agatagggct cattgaggtt ctgggaaaga tgcctgagca cttcatgggg 6480 aagacatggg aagcacttga caccatgtac gttgtggcca ctgcagagaa aggaggaaga 6540 gctcacagaa tggccctgga ggaactgcca gatgctcttc agacaattgc cttgattgcc 6600 ttattgagtg tgatgaccat gggagtattc ttcctcctca tgcagcggaa gggcattgga 6660 aagataggtt tgggaggcgc tgtcttggga gtagcgacct ttttctgttg gatggctgaa 6720 gttccaggaa cgaagatcgc cggaatgttg ctgctctccc ttctcttgat gattgtgcta 6780 attcctgagc cagagaagca acgttcgcag acagacaacc agctagccgt gttcctgatt 6840 tgtgtcatga cccttgtgag cgcagtggca gccaacgaga tgggttggct agataagacc 6900 aagagtgaca taagcagttt gtttgggcaa agaattgagg tcaaggagaa tttcagcatg 6960 ggagagtttc ttctggactt gaggccggca acagcctggt cactgtacgc tgtgacaaca 7020 gcggtcctca ctccactgct aaagcatttg atcacgtcag attacatcaa cacctcattg 7080 acctcaataa acgttcaggc aagtgcacta ttcacactcg cgcgaggctt ccccttcgtc 7140 gatgttggag tgtcggctct cctgctagca gccggatgct ggggacaagt caccctcacc 7200 gttacggtaa cagcggcaac actccttttt tgccactatg cctacatggt tcccggttgg 7260 caagctgagg caatgcgctc agcccagcgg cggacagcgg ccggaatcat gaagaacgct 7320 gtagtggatg gcatcgtggc cacggacgtc ccagaattag agcgcaccac acccatcatg 7380 cagaagaaag ttggacagat catgctgatc ttggtgtctc tagctgcagt agtagtgaac 7440 ccgtctgtga agacagtacg agaagccgga attttgatca cggccgcagc ggtgacgctt 7500 tgggagaatg gagcaagctc tgtttggaac gcaacaactg ccatcggact ctgccacatc 7560 atgcgtgggg gttggttgtc atgtctatcc ataacatgga cactcataaa gaacatggaa 7620 aaaccagggc taaaaagagg tggggcaaaa ggacgcacct tgggagaggt ttggaaagaa 7680 agactcaacc agatgacaaa agaagagttc actaggtacc gcaaagaggc catcatcgaa 7740 gtcgatcgct cagcggcaaa acacgccagg aaagaaggca atgtcactgg agggcatcca 7800 gtctctaggg gcacagcaaa actgagatgg ctggtcgaac ggaggtttct cgaaccggtc 7860 ggaaaagtga ttgaccttgg atgtggaaga ggcggttggt gttactatat ggcaacccaa 7920 aaaagagtcc aagaagtcag agggtacaca aagggcggtc ccggacatga agagccccaa 7980 ctagtgcaaa gttatggatg gaacattgtc accatgaaga gtggagtgga tgtgttctac 8040 agaccttctg agtgttgtga caccctcctt tgtgacatcg gagagtcctc gtcaagtgct 8100 gaggttgaag agcataggac gattcgggtc cttgaaatgg ttgaggactg gctgcaccga 8160 gggccaaggg aattttgcgt gaaggtgctc tgcccctaca tgccgaaagt catagagaag 8220 atggagctgc tccaacgccg gtatgggggg ggactggtca gaaacccact ctcacggaat 8280 tccacgcacg agatgtattg ggtgagtcga gcttcaggca atgtggtaca ttcagtgaat 8340 atgaccagcc aggtgctcct aggaagaatg gaaaaaagga cctggaaggg accccaatac 8400 gaggaagatg taaacttggg aagtggaacc agggcggtgg gaaaacccct gctcaactca 8460 gacaccagta aaatcaagaa caggattgaa cgactcaggc gtgagtacag ttcgacgtgg 8520 caccacgatg agaaccaccc atatagaacc tggaactacc acggcagtta tgatgtgaag 8580 cccacaggct ccgccagttc gctggtcaat ggagtggtca ggctcctctc aaaaccatgg 8640 gacaccatca cgaatgttac caccatggcc atgactgaca ctactccctt cgggcagcag 8700 cgagtgttca aagagaaggt ggacacgaaa gctcctgaac cgccagaagg agtgaagtac 8760 gtgctcaacg agaccaccaa ctggttgtgg gcgtttttgg ccagagaaaa acgtcccaga 8820 atgtgctctc gagaggaatt cataagaaag gtcaacagca atgcagcttt gggtgccatg 8880 tttgaagagc agaatcaatg gaggagcgcc agagaagcag ttgaagatcc aaaattttgg 8940 gagatggtgg atgaggagcg cgaggcacat ctgcgggggg aatgtcacac ttgcatttac 9000 aacatgatgg gaaagagaga gaaaaaaccc ggagagttcg gaaaggccaa gggaagcaga 9060 gccatttggt tcatgtggct cggagctcgc tttctggagt tcgaggctct gggttttctc 9120 aatgaagacc actggcttgg aagaaagaac tcaggaggag gtgtcgaggg cttgggcctc 9180 caaaaactgg gttacatcct gcgtgaagtt ggcacccggc ctgggggcaa gatctatgct 9240 gatgacacag ctggctggga cacccgcatc acgagagctg acttggaaaa tgaagctaag 9300 gtgcttgagc tgcttgatgg ggaacatcgg cgtcttgcca gggccatcat tgagctcacc 9360 tatcgtcaca aagttgtgaa agtgatgcgc ccggctgctg atggaagaac cgtcatggat 9420 gttatctcca gagaagatca gagggggagt ggacaagttg tcacctacgc cctaaacact 9480 ttcaccaacc tggccgtcca gctggtgagg atgatggaag gggaaggagt gattggccca 9540 gatgatgtgg agaaactcac aaaagggaaa ggacccaaag tcaggacctg gctgtttgag 9600 aatggggaag aaagactcag ccgcatggct gtcagtggag atgactgtgt ggtaaagccc 9660 ctggacgatc gctttgccac ctcgctccac ttcctcaatg ctatgtcaaa ggttcgcaaa 9720 gacatccaag agtggaaacc gtcaactgga tggtatgatt ggcagcaggt tccattttgc 9780 tcaaaccatt tcactgaatt gatcatgaaa gatggaagaa cactggtggt tccatgccga 9840 ggacaggatg aattggtagg cagagctcgc atatctccag gggccggatg gaacgtccgc 9900 gacactgctt gtctggctaa gtcttatgcc cagatgtggc tgcttctgta cttccacaga 9960 agagacctgc ggctcatggc caacgccatt tgctccgctg tccctgtgaa ttgggtccct 10020 accggaagaa ccacgtggtc catccatgca ggaggagagt ggatgacaac agaggacatg 10080 ttggaggtct ggaaccgtgt ttggatagag gagaatgaat ggatggaaga caaaacccca 10140 gtggagaaat ggagtgacgt cccatattca ggaaaacgag aggacatctg gtgtggcagc 10200 ctgattggca caagagcccg agccacgtgg gcagaaaaca tccaggtggc tatcaaccaa 10260 gtcagagcaa tcatcggaga tgagaagtat gtggattaca tgagttcact aaagagatat 10320 gaagacacaa ctttggttga ggacacagta ctgtagatat ttaatcaatt gtaaatagac 10380 aatataagta tgcataaaag tgtagtttta tagtagtatt tagtggtgtt agtgtaaata 10440 gttaagaaaa ttttgaggag aaagtcaggc cgggaagttc ccgccaccgg aagttgagta 10500 gacggtgctg cctgcgactc aaccccagga ggactgggtg aacaaagccg cgaagtgatc 10560 catgtaagcc ctcagaaccg tctcggaagg aggaccccac atgttgtaac ttcaaagccc 10620 aatgtcagac cacgctacgg cgtgctactc tgcggagagt gcagtctgcg atagtgcccc 10680 aggaggactg ggttaacaaa ggcaaaccaa cgccccacgc ggccctagcc ccggtaatgg 10740 tgttaaccag ggcgaaagga ctagaggtta gaggagaccc cgcggtttaa agtgcacggc 10800 ccagcctggc tgaagctgta ggtcagggga aggactagag gttagtggag accccgtgcc 10860 acaaaacacc acaacaaaac agcatattga cacctgggat agactaggag atcttctgct 10920 ctgcacaacc agccacacgg cacag 10945 6 1542 DNA Escherichia coli 16S Ribosomal RNA misc_feature (896)..(896) n is a, c, g, or t 6 aaattgaaga gtttgatcat ggctcagatt gaacgctggc ggcaggccta acacatgcaa 60 gtcgaacggt aacaggaakc agcttgctga tttgctgacg agtggcggac gggtgagtaa 120 tgtctgggaa actgcctgat ggagggggat aactactgga aacggtagct aataccgcat 180 aacgtcgcaa gaccaaagag ggggaccttc gggcctcttg ccatcggatg tgcccagatg 240 ggattagcta gtaggtgggg taaaggctca cctaggcgac gatccctagc tggtctgaga 300 ggatgaccag ccacactgga actgagacac ggtccagact cctacgggag gcagcagtgg 360 ggaatattgc acaatgggcg caagcctgat gcagccatgc cgcgtgtatg aagaaggcct 420 tcgggttgta aagtactttc agcggggagg aagggagtaa agttaatacc tttgctcatt 480 gacgttaccc gcagaagaag caccggctaa ctccgtgcca gcagccgcgg taatacggag 540 ggtgcaagcg ttaatcggaa ttactgggcg taaagcgcac gcaggcggtt tgttaagtca 600 gatgtgaaat ccccgggctc aacctgggaa ctgcatctga tactggcaag cttgagtctc 660 gtagaggggg gtagaattcc aggtgtagcg gtgaaatgcg tagagatctg gaggaatacc 720 ggtggcgaag gcggccccct ggacgaagac tgacgctcag gtgcgaaagc gtggggagca 780 aacaggatta gataccctgg tagtccacgc cgtaaacgat gtcgacttgg aggttgtgcc 840 cttgaggcgt ggcttccgga gctaacgcgt taagtckacc gcctggggag tacggncgca 900 aggttaaaac tcaaatgaat tgacgggggc ccgcacaagc ggtggagcat gtggtttaat 960 tcgatgcaac gcgaagaacc ttacctggtc ttgacatcca cagaactttc cagagatgga 1020 ttggtgcctt cgggaactgt gagacaggtg ctgcatggct gtcgtcagct cgtgttgtga 1080 aatgttgggt taagtcccgc aacgagcgca acccttatcc tttgttgcca gcggtccggc 1140 cgggaactca aaggagactg ccagtgataa actggaggaa ggtggggatg acgtcaagtc 1200 atcatggccc ttacgaccag ggctacacac gtgctacaat ggcgcataca aagagaagcg 1260 ayctcgcgag agcaagcgga cctcataaag tgcgtcgtag tccggattgg agtctgcaac 1320 tcgactccat gaagtcggaa tcgctagtaa tcgtggatca gaatgccacg gtgaatacgt 1380 tcccgggcct tgtacacacc gcccgtcaca ccatgggagt gggttgcaaa agaagtaggt 1440 agcttaacct tcgggagggc gcttaccact ttgtgattca tgactggggt gaagtcgtaa 1500 caaggtaacc gtaggggaac ctgcggttgg atcacctcct ta 1542 7 2905 DNA Escherichia coli 23 S Ribosomal RNA 7 ggttaagcga ctaagcgtac acggtggatg ccctggcagt cagaggcgat gaaggacgtg 60 ctaatctgcg ataagcgtcg gtaaggtgat atgaaccgtt ataaccggcg atttccgaat 120 ggggaaaccc agtgtgtttc gacacactat cattaactga atccataggt taatgaggcg 180 aaccggggga actgaaacat ctaagtaccc cgaggaaaag aaatcaaccg agattccccc 240 agtagcggcg agcgaacggg gagcagccca gagcctgaat cagtgtgtgt gttagtggaa 300 gcgtctggaa aggcgcgcga tacagggtga cagccccgta cacaaaaatg cacatgctgt 360 gagctcgatg agtagggcgg gacacgtggt atcctgtctg aatatggggg gaccatcctc 420 caaggctaaa tactcctgac tgaccgatag tgaaccagta ccgtgaggga aaggcgaaaa 480 gaaccccggc gaggggagtg aaaaagaacc tgaaaccgtg tacgtacaag cagtgggagc 540 acgcttaggc gtgtgactgc gtaccttttg tataatgggt cagcgactta tattctgtag 600 caaggttaac cgaatagggg agccgaaggg aaaccgagtc ttaactgggc gttaagttgc 660 agggtataga cccgaaaccc ggtgatctag ccatgggcag gttgaaggtt gggtaacact 720 aactggagga ccgaaccgac taatgttgaa aaattagcgg atgacttgtg gctgggggtg 780 aaaggccaat caaaccggga gatagctggt tctccccgaa agctatttag gtagcgcctc 840 gtgaattcat ctccgggggt agagcactgt ttcggcaagg gggtcatccc gacttaccaa 900 cccgatgcaa actgcgaata ccggagaatg ttatcacggg agacacacgg cgggtgctaa 960 cgtccgtcgt gaagagggaa acaacccaga ccgccagcta aggtcccaaa gtcatggtta 1020 agtgggaaac gatgtgggaa ggcccagaca gccaggatgt tggcttagaa gcagccatca 1080 tttaaagaaa gcgtaatagc tcactggtcg agtcggcctg cgcggaagat gtaacggggc 1140 taaaccatgc accgaagctg cggcagcgac gcttatgcgt tgttgggtag gggagcgttc 1200 tgtaagcctg cgaaggtgtg ctgtgaggca tgctggaggt atcagaagtg cgaatgctga 1260 cataagtaac gataaagcgg gtgaaaagcc cgctcgccgg aagaccaagg gttcctgtcc 1320 aacgttaatc ggggcagggt gagtcgaccc ctaaggcgag gccgaaaggc gtagtcgatg 1380 ggaaacaggt taatattcct gtacttggtg ttactgcgaa ggggggacgg agaaggctat 1440 gttggccggg cgacggttgt cccggtttaa gcgtgtaggc tggttttcca ggcaaatccg 1500 gaaaatcaag gctgaggcgt gatgacgagg cactacggtg ctgaagcaac aaatgccctg 1560 cttccaggaa aagcctctaa gcatcaggta acatcaaatc gtaccccaaa ccgacacagg 1620 tggtcaggta gagaatacca aggcgcttga gagaactcgg gtgaaggaac taggcaaaat 1680 ggtgccgtaa cttcgggaga aggcacgctg atatgtaggt gaagcgactt gctcgtggag 1740 ctgaaatcag tcgaagatac cagctggctg caactgttta ttaaaaacac agcactgtgc 1800 aaacacgaaa gtggacgtat acggtgtgac gcctgcccgg tgccggaagg ttaattgatg 1860 gggttagcgg taacgcgaag ctcttgatcg aagccccggt aaacggcggc cgtaactata 1920 acggtcctaa ggtagcgaaa ttccttgtcg ggtaagttcc gacctgcacg aatggcgtaa 1980 tgatggccag gctgtctcca cccgagactc agtgaaattg aactcgctgt gaagatgcag 2040 tgtacccgcg gcaagacgga aagaccccgt gaacctttac tatagcttga cactgaacat 2100 tgagccttga tgtgtaggat aggtgggagg ctttgaagtg tggacgccag tctgcatgga 2160 gccgaccttg aaataccacc ctttaatgtt tgatgttcta acgttggccc gtaatccggg 2220 ttgcggacag tgtctggtgg gtagtttgac tggggcggtc tcctcctaaa gagtaacgga 2280 ggagcacgaa ggttggctaa tcctggtcgg acatcaggag gttagtgcaa tggcataagc 2340 cagcttgact gcgagcgtga cggcgcgagc aggtgcgaaa gcaggtcata gtgatccggt 2400 ggttctgaat ggaagggcca tcgctcaacg gataaaaggt actccgggga taacaggctg 2460 ataccgccca agagttcata tcgacggcgg tgtttggcac ctcgatgtcg gctcatcaca 2520 tcctggggct gaagtaggtc ccaagggtat ggctgttcgc catttaaagt ggtacgcgag 2580 ctgggtttag aacgtcgtga gacagttcgg tccctatctg ccgtgggcgc tggagaactg 2640 aggggggctg ctcctagtac gagaggaccg gagtggacgc atcactggtg ttcgggttgt 2700 catgccaatg cactgcccgg tagctaaatg cggaagagat aagtgctgaa agcatctaag 2760 cacgaaactt gccccgagat gagttctccc tgacccttta agggtcctga aggaacgttg 2820 aagacgacga cgttgatagg ccgggtgtgt aagcgcagcg atgcgttgag ctaaccggta 2880 ctaatgaacc gtgaggctta acctt 2905 8 1798 DNA Yeast (S. cerevisiae) 8 tatctggttg atcctgccag tagtcatatg cttgtctcaa agattaagcc atgcatgtct 60 aagtataagc aatttataca gtgaaactgc gaatggctca ttaaatcagt tatcgtttat 120 ttgatagttc ctttactaca tggtataacc gtggtaattc tagagctaat acatgcttaa 180 aatctcgacc ctttggaaga gatgtattta ttagataaaa aatcaatgtc ttcggactct 240 ttgatgattc ataataactt ttcgaatcgc atggccttgt gctggcgatg gttcattcaa 300 atttctgccc tatcaacttt cgatggtagg atagtggcct accatggttt caacgggtaa 360 cggggaataa gggttcgatt ccggagaggg agcctgagaa acggctacca catccaagga 420 aggcagcagg cgcgcaaatt acccaatcct aattcaggga ggtagtgaca ataaataacg 480 atacagggcc cattcgggtc ttgtaattgg aatgagtaca atgtaaatac cttaacgagg 540 aacaattgga gggcaagtct ggtgccagca gccgcggtaa ttccagctcc aatagcgtat 600 attaaagttg ttgcagttaa aaagctcgta gttgaacttt gggcccggtt ggccggtccg 660 attttttcgt gtactggatt tccaacgggg cctttccttc tggctaacct tgagtccttg 720 tggctcttgg cgaaccagga cttttacttt gaaaaaatta gagtgttcaa agcaggcgta 780 ttgctcgaat atattagcat ggaataatag aataggacgt ttggttctat tttgttggtt 840 tctaggacca tcgtaatgat taatagggac ggtcgggggc atcggtattc aattgtcgag 900 gtgaaattct tggatttatt gaagactaac tactgcgaaa gcatttgcca aggacgtttt 960 cattaatcaa gaacgaaagt taggggatcg aagatgatct ggtaccgtcg tagtcttaac 1020 cataaactat gccgactaga tcgggtggtg tttttttaat gacccactcg gtaccttacg 1080 agaaatcaaa gtctttgggt tctgggggga gtatggtcgc aaggctgaaa cttaaaggaa 1140 ttgacggaag ggcaccacta ggagtggagc ctgcggctaa tttgactcaa cacggggaaa 1200 ctcaccaggt ccagacacaa taaggattga cagattgaga gctctttctt gattttgtgg 1260 gtggtggtgc atggccgttt ctcagttggt ggagtgattt gtctgcttaa ttgcgataac 1320 gaacgagacc ttaacctact aaatagtggt gctagcattt gctggttatc cacttcttag 1380 agggactatc ggtttcaagc cgatggaagt ttgaggcaat aacaggtctg tgatgccctt 1440 agaacgttct gggccgcacg cgcgctacac tgacggagcc agcgagtcta accttggccg 1500 agaggtcttg gtaatcttgt gaaactccgt cgtgctgggg atagagcatt gtaattattg 1560 ctcttcaacg aggaattcct agtaagcgca agtcatcagc ttgcgttgat tacgtccctg 1620 ccctttgtac acaccgcccg tcgctagtac cgattgaatg gcttagtgag gcctcaggat 1680 ctgcttagag aagggggcaa ctccatctca gagcggagaa tttggacaaa cttggtcatt 1740 tagaggaact aaaagtcgta acaaggtttc cgtaggtgaa cctgcggaag gatcatta 1798 9 3911 DNA Yeast 25S Ribosomal RNA 9 aattccgtga tgggccttta ggttttacca actgcggcta atcttttttt atactgagcg 60 tattggaacg ttatcgataa gaagagagcg tctaggcgaa caatgttctt aaagtttgac 120 ctcaaatcag gtaggagtac ccgctgaact taagcatatc aataagcgga ggaaaagaaa 180 ccaaccggat tgccttagta acggcgagtg aagcggcaaa agctcaaatt tgaaatctgg 240 taccttcggt gcccgagttg taatttggag agggcaactt tggggccgtt ccttgtctat 300 gttccttgga acaggacgtc atagagggtg agcatcccgt gtggcgagga gtgcggttct 360 ttgtaaagtg ccttcgaaga gtcgagttgt ttgggaatgc agctctaagt gggtggtaaa 420 ttccatctaa agctaaatat tggcgagaga ccgatagcga acaagtacag tgatggaaag 480 atgaaaagaa ctttgaaaag agagtgaaaa agtacgtgaa attgttgaaa gggaagggca 540 tttgatcaga catggtgttt tgtgccctct gctccttgtg ggtaggggaa tctcgcattt 600 cactgggcca gcatcagttt tggtggcagg ataaatccat aggaatgtag cttgcctcgg 660 taagtattat agcctgtggg aatactgcca gctgggactg aggactgcga cgtaagtcaa 720 ggatgctggc ataatggtta tatgccgccc gtcttgaaac acggaccaag gagtctaacg 780 tctatgcgag tgtttgggtg taaaacccat acgcgtaatg aaagtgaacg taggttgggg 840 cctcgcaaga ggtgcacaat cgaccgatcc tgatgtcttc ggatggattt gagtaagagc 900 atagctgttg ggacccgaaa gatggtgaac tatgcctgaa tagggtgaag ccagaggaaa 960 ctctggtgga ggctcgtagc ggttctgacg tgcaaatcga tcgtcgaatt tgggtatagg 1020 ggcgaaagac taatcgaacc atctagtagc tggttcctgc cgaagtttcc ctcaggatag 1080 cagaagctcg tatcagtttt atgaggtaaa gcgaatgatt agaggttccg gggtcgaaat 1140 gaccttgacc tattctcaaa ctttaaatat gtaagaagtc cttgttactt aattgaacgt 1200 ggacatttga atgaagagct tttagtgggc catttttggt aagcagaact ggcgatgcgg 1260 gatgaaccga acgtagagtt aaggtgccgg aatacacgct catcagacac cacaaaaggt 1320 gttagttcat ctagacagcc ggacggtggc catggaagtc ggaatccgct aaggagtgtg 1380 taacaactca ccggccgaat gaactagccc tgaaaatgga tggcgctcaa gcgtgttacc 1440 tatactctac cgtcagggtt gatatgatgc cctgacgagt aggcaggcgt ggaggtcagt 1500 gacgaagcct agaccgtaag gtcgggtcga acggcctcta gtgcagatct tggtggtagt 1560 agcaaatatt caaatgagaa ctttgaagac tgaagtgggg aaaggttcca cgtcaacagc 1620 agttggacgt gggttagtcg atcctaagag atggggaagc tccgtttcaa aggcctgatt 1680 ttatgcaggc caccatcgaa agggaatccg gtaagattcc ggaacttgga tatggattct 1740 tcacggtaac gtaactgaat gtggagacgt cggcgcgagc cctgggagga gttatctttt 1800 cttcttaaca gcttatcacc ccggaattgg tttatccgga gatggggtct tatggctgga 1860 agaggccagc acctttgctg gctccggtgc gcttgtgacg gcccgtgaaa atccacagga 1920 aggaatagtt ttcatgctag gtcgtactga taaccgcagc aggtctccaa ggtgaacagc 1980 ctctagttga tagaataatg tagataaggg aagtcggcaa aatagatccg taacttcggg 2040 ataaggattg gctctaaggg tcgggtagtg agggccttgg tcagacgcag cgggcgtgct 2100 tgtggactgc ttggtggggc ttgctctgct aggcggacta cttgcgtgcc ttgttgtaga 2160 cggccttggt aggtctcttg tagaccgtcg cttgctacaa ttaacagatc aacttagaac 2220 tggtacggac aaggggaatc tgactgtcta attaaaacat agcattgcga tggtcagaaa 2280 gtgatgttga cgcaatgtga tttctgccca gtgctctgaa tgtcaaagtg aagaaattca 2340 accaagcgcg agtaaacggc gggagtaact atgactctct taaggtagcc aaatgcctcg 2400 tcatctaatt agtgacgcgc atgaatggat taacgagatt cccactgtcc ctatctacta 2460 tctagcgaaa ccacagccaa gggaacgggc ttggcagaat cagcggggaa agaagaccct 2520 gttgagcttg actctagttt gacattgtga agagacatag agggtgtaga ataagtggga 2580 gcttcggcgc cagtgaaata ccactacctt tatagtttct ttacttattc aatgaagcgg 2640 agctggaatt cattttccac gttctagcat tcaaggtccc attcggggct gatccgggtt 2700 gaagacattg tcaggtgggg agtttggctg gggcggcaca tctgttaaac gataacgcag 2760 atgtcctaag gggggctcat ggagaacaga aatctccagt agaacaaaag ggtaaagccc 2820 cttagtttga tttcagtgtg aatacaaacc attgaaagtg tggcctatcg atcctttagt 2880 ccctcggaat ttgaggctag aggtgccaga aaagttacca cagggataac tggcttgtgg 2940 cagtcaagcg ttcatagcga cattgctttt tgattcttcg atgtcggctc ttcctatcat 3000 accgaagcag aattcggtaa gcgttggatt gttcacccac taatagggaa catgagctgg 3060 gtttagaccg tcgtgagaca ggttagtttt accctactga tgaatgttac cagcaatagt 3120 aattgaactt agtacgagag gaacagttca ttcggataat tggtttttgc ggctgtctga 3180 tcaggcattg ccgcgaagca ccatccgctg gattatggct gaacgcctct aagtcagaat 3240 ccatgctaga acgcggtgat ttctttgctc cacacaatat agatggatac gaataaggcg 3300 tccttgtggc gtcgctgaac catagcaggc tagcaacggt gcacttggcg gaaaggcctt 3360 gggtgcttgc tggcgaattg caatgtcatt ttgcgtgggg ataaatcatt tgtatacgac 3420 ttagatgtac aacggggtat tgtaagcggt agagtagcct tgttgttacg atctgctgag 3480 attaagcctt tgttgtctga tttgtttttt atttctttct aagtgggtac tggcaggagc 3540 cggggcctag tttagagaga agtagactca acaagtctct ataaatttta tttgtcttaa 3600 gaattctatg atccgggtaa aaacatgtat tgtatatatc tattataata tacgatgagg 3660 atgatagtgt gtaagagtgt accatttact aatgtatgta agttactatt tactatttgg 3720 tctttttatt ttttattttt tttttttttt tcgttgcaaa gatgggttga aagagaaggg 3780 ctttcacaaa gcttcccgag cgtgaaagga tttgcccgga cagtttgctt catggagcag 3840 ttttttccgc accatcagag cggcaaacat gagtgcttgt ataagtttag agaattgaga 3900 aaagctcatt t 3911 10 16569 DNA Human mitochondrial DNA 10 gatcacaggt ctatcaccct attaaccact cacgggagct ctccatgcat ttggtatttt 60 cgtctggggg gtatgcacgc gatagcattg cgagacgctg gagccggagc accctatgtc 120 gcagtatctg tctttgattc ctgcctcatc ctattattta tcgcacctac gttcaatatt 180 acaggcgaac atacttacta aagtgtgtta attaattaat gcttgtagga cataataata 240 acaattgaat gtctgcacag ccactttcca cacagacatc ataacaaaaa atttccacca 300 aaccccccct cccccgcttc tggccacagc acttaaacac atctctgcca aaccccaaaa 360 acaaagaacc ctaacaccag cctaaccaga tttcaaattt tatcttttgg cggtatgcac 420 ttttaacagt caccccccaa ctaacacatt attttcccct cccactccca tactactaat 480 ctcatcaata caacccccgc ccatcctacc cagcacacac acaccgctgc taaccccata 540 ccccgaacca accaaacccc aaagacaccc cccacagttt atgtagctta cctcctcaaa 600 gcaatacact gaaaatgttt agacgggctc acatcacccc ataaacaaat aggtttggtc 660 ctagcctttc tattagctct tagtaagatt acacatgcaa gcatccccgt tccagtgagt 720 tcaccctcta aatcaccacg atcaaaaggg acaagcatca agcacgcagc aatgcagctc 780 aaaacgctta gcctagccac acccccacgg gaaacagcag tgattaacct ttagcaataa 840 acgaaagttt aactaagcta tactaacccc agggttggtc aatttcgtgc cagccaccgc 900 ggtcacacga ttaacccaag tcaatagaag ccggcgtaaa gagtgtttta gatcaccccc 960 tccccaataa agctaaaact cacctgagtt gtaaaaaact ccagttgaca caaaatagac 1020 tacgaaagtg gctttaacat atctgaacac acaatagcta agacccaaac tgggattaga 1080 taccccacta tgcttagccc taaacctcaa cagttaaatc aacaaaactg ctcgccagaa 1140 cactacgagc cacagcttaa aactcaaagg acctggcggt gcttcatatc cctctagagg 1200 agcctgttct gtaatcgata aaccccgatc aacctcacca cctcttgctc agcctatata 1260 ccgccatctt cagcaaaccc tgatgaaggc tacaaagtaa gcgcaagtac ccacgtaaag 1320 acgttaggtc aaggtgtagc ccatgaggtg gcaagaaatg ggctacattt tctaccccag 1380 aaaactacga tagcccttat gaaacttaag ggtcgaaggt ggatttagca gtaaactaag 1440 agtagagtgc ttagttgaac agggccctga agcgcgtaca caccgcccgt caccctcctc 1500 aagtatactt caaaggacat ttaactaaaa cccctacgca tttatataga ggagacaagt 1560 cgtaacatgg taagtgtact ggaaagtgca cttggacgaa ccagagtgta gcttaacaca 1620 aagcacccaa cttacactta ggagatttca acttaacttg accgctctga gctaaaccta 1680 gccccaaacc cactccacct tactaccaga caaccttagc caaaccattt acccaaataa 1740 agtataggcg atagaaattg aaacctggcg caatagatat agtaccgcaa gggaaagatg 1800 aaaaattata accaagcata atatagcaag gactaacccc tataccttct gcataatgaa 1860 ttaactagaa ataactttgc aaggagagcc aaagctaaga cccccgaaac cagacgagct 1920 acctaagaac agctaaaaga gcacacccgt ctatgtagca aaatagtggg aagatttata 1980 ggtagaggcg acaaacctac cgagcctggt gatagctggt tgtccaagat agaatcttag 2040 ttcaacttta aatttgccca cagaaccctc taaatcccct tgtaaattta actgttagtc 2100 caaagaggaa cagctctttg gacactagga aaaaaccttg tagagagagt aaaaaattta 2160 acacccatag taggcctaaa agcagccacc aattaagaaa gcgttcaagc tcaacaccca 2220 ctacctaaaa aatcccaaac atataactga actcctcaca cccaattgga ccaatctatc 2280 accctataga agaactaatg ttagtataag taacatgaaa acattctcct ccgcataagc 2340 ctgcgtcaga ttaaaacact gaactgacaa ttaacagccc aatatctaca atcaaccaac 2400 aagtcattat taccctcact gtcaacccaa cacaggcatg ctcataagga aaggttaaaa 2460 aaagtaaaag gaactcggca aatcttaccc cgcctgttta ccaaaaacat cacctctagc 2520 atcaccagta ttagaggcac cgcctgccca gtgacacatg tttaacggcc gcggtaccct 2580 aaccgtgcaa aggtagcata atcacttgtt ccttaaatag ggacctgtat gaatggctcc 2640 acgagggttc agctgtctct tacttttaac cagtgaaatt gacctgcccg tgaagaggcg 2700 ggcataacac agcaagacga gaagacccta tggagcttta atttattaat gcaaacagta 2760 cctaacaaac ccacaggtcc taaactacca aacctgcatt aaaaatttcg gttggggcga 2820 cctcggagca gaacccaacc tccgagcagt acatgctaag acttcaccag tcaaagcgaa 2880 ctactatact caattgatcc aataacttga ccaacggaac aagttaccct agggataaca 2940 gcgcaatcct attctagagt ccatatcaac aatagggttt acgacctcga tgttggatca 3000 ggacatcccg atggtgcagc cgctattaaa ggttcgtttg ttcaacgatt aaagtcctac 3060 gtgatctgag ttcagaccgg agtaatccag gtcggtttct atctaccttc aaattcctcc 3120 ctgtacgaaa ggacaagaga aataaggcct acttcacaaa gcgccttccc ccgtaaatga 3180 tatcatctca acttagtatt atacccacac ccacccaaga acagggtttg ttaagatggc 3240 agagcccggt aatcgcataa aacttaaaac tttacagtca gaggttcaat tcctcttctt 3300 aacaacatac ccatggccaa cctcctactc ctcattgtac ccattctaat cgcaatggca 3360 ttcctaatgc ttaccgaacg aaaaattcta ggctatatac aactacgcaa aggccccaac 3420 gttgtaggcc cctacgggct actacaaccc ttcgctgacg ccataaaact cttcaccaaa 3480 gagcccctaa aacccgccac atctaccatc accctctaca tcaccgcccc gaccttagct 3540 ctcaccatcg ctcttctact atgaaccccc ctccccatac ccaaccccct ggtcaacctc 3600 aacctaggcc tcctatttat tctagccacc tctagcctag ccgtttactc aatcctctga 3660 tcagggtgag catcaaactc aaactacgcc ctgatcggcg cactgcgagc agtagcccaa 3720 acaatctcat atgaagtcac cctagccatc attctactat caacattact aataagtggc 3780 tcctttaacc tctccaccct tatcacaaca caagaacacc tctgattact cctgccatca 3840 tgacccttgg ccataatatg atttatctcc acactagcag agaccaaccg aacccccttc 3900 gaccttgccg aaggggagtc cgaactagtc tcaggcttca acatcgaata cgccgcaggc 3960 cccttcgccc tattcttcat agccgaatac acaaacatta ttataataaa caccctcacc 4020 actacaatct tcctaggaac aacatatgac gcactctccc ctgaactcta cacaacatat 4080 tttgtcacca agaccctact tctaacctcc ctgttcttat gaattcgaac agcatacccc 4140 cgattccgct acgaccaact catacacctc ctatgaaaaa acttcctacc actcacccta 4200 gcattactta tatgatatgt ctccataccc attacaatct ccagcattcc ccctcaaacc 4260 taagaaatat gtctgataaa agagttactt tgatagagta aataatagga gcttaaaccc 4320 ccttatttct aggactatga gaatcgaacc catccctgag aatccaaaat tctccgtgcc 4380 acctatcaca ccccatccta aagtaaggtc agctaaataa gctatcgggc ccataccccg 4440 aaaatgttgg ttataccctt cccgtactaa ttaatcccct ggcccaaccc gtcatctact 4500 ctaccatctt tgcaggcaca ctcatcacag cgctaagctc gcactgattt tttacctgag 4560 taggcctaga aataaacatg ctagctttta ttccagttct aaccaaaaaa ataaaccctc 4620 gttccacaga agctgccatc aagtatttcc tcacgcaagc aaccgcatcc ataatccttc 4680 taatagctat cctcttcaac aatatactct ccggacaatg aaccataacc aatactacca 4740 atcaatactc atcattaata atcataatag ctatagcaat aaaactagga atagccccct 4800 ttcacttctg agtcccagag gttacccaag gcacccctct gacatccggc ctgcttcttc 4860 tcacatgaca aaaactagcc cccatctcaa tcatatacca aatctctccc tcactaaacg 4920 taagccttct cctcactctc tcaatcttat ccatcatagc aggcagttga ggtggattaa 4980 accagaccca gctacgcaaa atcttagcat actcctcaat tacccacata ggatgaataa 5040 tagcagttct accgtacaac cctaacataa ccattcttaa tttaactatt tatattatcc 5100 taactactac cgcattccta ctactcaact taaactccag caccacgacc ctactactat 5160 ctcgcacctg aaacaagcta acatgactaa cacccttaat tccatccacc ctcctctccc 5220 taggaggcct gcccccgcta accggctttt tgcccaaatg ggccattatc gaagaattca 5280 caaaaaacaa tagcctcatc atccccacca tcatagccac catcaccctc cttaacctct 5340 acttctacct acgcctaatc tactccacct caatcacact actccccata tctaacaacg 5400 taaaaataaa atgacagttt gaacatacaa aacccacccc attcctcccc acactcatcg 5460 cccttaccac gctactccta cctatctccc cttttatact aataatctta tagaaattta 5520 ggttaaatac agaccaagag ccttcaaagc cctcagtaag ttgcaatact taatttctgt 5580 aacagctaag gactgcaaaa ccccactctg catcaactga acgcaaatca gccactttaa 5640 ttaagctaag cccttactag accaatggga cttaaaccca caaacactta gttaacagct 5700 aagcacccta atcaactggc ttcaatctac ttctcccgcc gccgggaaaa aaggcgggag 5760 aagccccggc aggtttgaag ctgcttcttc gaatttgcaa ttcaatatga aaatcacctc 5820 ggagctggta aaaagaggcc taacccctgt ctttagattt acagtccaat gcttcactca 5880 gccattttac ctcaccccca ctgatgttcg ccgaccgttg actattctct acaaaccaca 5940 aagacattgg aacactatac ctattattcg gcgcatgagc tggagtccta ggcacagctc 6000 taagcctcct tattcgagcc gagctgggcc agccaggcaa ccttctaggt aacgaccaca 6060 tctacaacgt tatcgtcaca gcccatgcat ttgtaataat cttcttcata gtaataccca 6120 tcataatcgg aggctttggc aactgactag ttcccctaat aatcggtgcc cccgatatgg 6180 cgtttccccg cataaacaac ataagcttct gactcttacc tccctctctc ctactcctgc 6240 tcgcatctgc tatagtggag gccggagcag gaacaggttg aacagtctac cctcccttag 6300 cagggaacta ctcccaccct ggagcctccg tagacctaac catcttctcc ttacacctag 6360 caggtgtctc ctctatctta ggggccatca atttcatcac aacaattatc aatataaaac 6420 cccctgccat aacccaatac caaacgcccc tcttcgtctg atccgtccta atcacagcag 6480 tcctacttct cctatctctc ccagtcctag ctgctggcat cactatacta ctaacagacc 6540 gcaacctcaa caccaccttc ttcgaccccg ccggaggagg agaccccatt ctataccaac 6600 acctattctg atttttcggt caccctgaag tttatattct tatcctacca ggcttcggaa 6660 taatctccca tattgtaact tactactccg gaaaaaaaga accatttgga tacataggta 6720 tggtctgagc tatgatatca attggcttcc tagggtttat cgtgtgagca caccatatat 6780 ttacagtagg aatagacgta gacacacgag catatttcac ctccgctacc ataatcatcg 6840 ctatccccac cggcgtcaaa gtatttagct gactcgccac actccacgga agcaatatga 6900 aatgatctgc tgcagtgctc tgagccctag gattcatctt tcttttcacc gtaggtggcc 6960 tgactggcat tgtattagca aactcatcac tagacatcgt actacacgac acgtactacg 7020 ttgtagccca cttccactat gtcctatcaa taggagctgt atttgccatc ataggaggct 7080 tcattcactg atttccccta ttctcaggct acaccctaga ccaaacctac gccaaaatcc 7140 atttcactat catattcatc ggcgtaaatc taactttctt cccacaacac tttctcggcc 7200 tatccggaat gccccgacgt tactcggact accccgatgc atacaccaca tgaaacatcc 7260 tatcatctgt aggctcattc atttctctaa cagcagtaat attaataatt ttcatgattt 7320 gagaagcctt cgcttcgaag cgaaaagtcc taatagtaga agaaccctcc ataaacctgg 7380 agtgactata tggatgcccc ccaccctacc acacattcga agaacccgta tacataaaat 7440 ctagacaaaa aaggaaggaa tcgaaccccc caaagctggt ttcaagccaa ccccatggcc 7500 tccatgactt tttcaaaaag gtattagaaa aaccatttca taactttgtc aaagttaaat 7560 tataggctaa atcctatata tcttaatggc acatgcagcg caagtaggtc tacaagacgc 7620 tacttcccct atcatagaag agcttatcac ctttcatgat cacgccctca taatcatttt 7680 ccttatctgc ttcctagtcc tgtatgccct tttcctaaca ctcacaacaa aactaactaa 7740 tactaacatc tcagacgctc aggaaataga aaccgtctga actatcctgc ccgccatcat 7800 cctagtcctc atcgccctcc catccctacg catcctttac ataacagacg aggtcaacga 7860 tccctccctt accatcaaat caattggcca ccaatggtac tgaacctacg agtacaccga 7920 ctacggcgga ctaatcttca actcctacat acttccccca ttattcctag aaccaggcga 7980 cctgcgactc cttgacgttg acaatcgagt agtactcccg attgaagccc ccattcgtat 8040 aataattaca tcacaagacg tcttgcactc atgagctgtc cccacattag gcttaaaaac 8100 agatgcaatt cccggacgtc taaaccaaac cactttcacc gctacacgac cgggggtata 8160 ctacggtcaa tgctctgaaa tctgtggagc aaaccacagt ttcatgccca tcgtcctaga 8220 attaattccc ctaaaaatct ttgaaatagg gcccgtattt accctatagc accccctcta 8280 ccccctctag agcccactgt aaagctaact tagcattaac cttttaagtt aaagattaag 8340 agaaccaaca cctctttaca gtgaaatgcc ccaactaaat actaccgtat ggcccaccat 8400 aattaccccc atactcctta cactattcct catcacccaa ctaaaaatat taaacacaaa 8460 ctaccaccta cctccctcac caaagcccat aaaaataaaa aattataaca aaccctgaga 8520 accaaaatga acgaaaatct gttcgcttca ttcattgccc ccacaatcct aggcctaccc 8580 gccgcagtac tgatcattct atttccccct ctattgatcc ccacctccaa atatctcatc 8640 aacaaccgac taatcaccac ccaacaatga ctaatcaaac taacctcaaa acaaatgata 8700 accatacaca acactaaagg acgaacctga tctcttatac tagtatcctt aatcattttt 8760 attgccacaa ctaacctcct cggactcctg cctcactcat ttacaccaac cacccaacta 8820 tctataaacc tagccatggc catcccctta tgagcgggca cagtgattat aggctttcgc 8880 tctaagatta aaaatgccct agcccacttc ttaccacaag gcacacctac accccttatc 8940 cccatactag ttattatcga aaccatcagc ctactcattc aaccaatagc cctggccgta 9000 cgcctaaccg ctaacattac tgcaggccac ctactcatgc acctaattgg aagcgccacc 9060 ctagcaatat caaccattaa ccttccctct acacttatca tcttcacaat tctaattcta 9120 ctgactatcc tagaaatcgc tgtcgcctta atccaagcct acgttttcac acttctagta 9180 agcctctacc tgcacgacaa cacataatga cccaccaatc acatgcctat catatagtaa 9240 aacccagccc atgaccccta acaggggccc tctcagccct cctaatgacc tccggcctag 9300 ccatgtgatt tcacttccac tccataacgc tcctcatact aggcctacta accaacacac 9360 taaccatata ccaatgatgg cgcgatgtaa cacgagaaag cacataccaa ggccaccaca 9420 caccacctgt ccaaaaaggc cttcgatacg ggataatcct atttattacc tcagaagttt 9480 ttttcttcgc aggatttttc tgagcctttt accactccag cctagcccct accccccaat 9540 taggagggca ctggccccca acaggcatca ccccgctaaa tcccctagaa gtcccactcc 9600 taaacacatc cgtattactc gcatcaggag tatcaatcac ctgagctcac catagtctaa 9660 tagaaaacaa ccgaaaccaa ataattcaag cactgcttat tacaatttta ctgggtctct 9720 attttaccct cctacaagcc tcagagtact tcgagtctcc cttcaccatt tccgacggca 9780 tctacggctc aacatttttt gtagccacag gcttccacgg acttcacgtc attattggct 9840 caactttcct cactatctgc ttcatccgcc aactaatatt tcactttaca tccaaacatc 9900 actttggctt cgaagccgcc gcctgatact ggcattttgt agatgtggtt tgactatttc 9960 tgtatgtctc catctattga tgagggtctt actcttttag tataaatagt accgttaact 10020 tccaattaac tagttttgac aacattcaaa aaagagtaat aaacttcgcc ttaattttaa 10080 taatcaacac cctcctagcc ttactactaa taattattac attttgacta ccacaactca 10140 acggctacat agaaaaatcc accccttacg agtgcggctt cgaccctata tcccccgccc 10200 gcgtcccttt ctccataaaa ttcttcttag tagctattac cttcttatta tttgatctag 10260 aaattgccct ccttttaccc ctaccatgag ccctacaaac aactaacctg ccactaatag 10320 ttatgtcatc cctcttatta atcatcatcc tagccctaag tctggcctat gagtgactac 10380 aaaaaggatt agactgaacc gaattggtat atagtttaaa caaaacgaat gatttcgact 10440 cattaaatta tgataatcat atttaccaaa tgcccctcat ttacataaat attatactag 10500 catttaccat ctcacttcta ggaatactag tatatcgctc acacctcata tcctccctac 10560 tatgcctaga aggaataata ctatcgctgt tcattatagc tactctcata accctcaaca 10620 cccactccct cttagccaat attgtgccta ttgccatact agtctttgcc gcctgcgaag 10680 cagcggtggg cctagcccta ctagtctcaa tctccaacac atatggccta gactacgtac 10740 ataacctaaa cctactccaa tgctaaaact aatcgtccca acaattatat tactaccact 10800 gacatgactt tccaaaaaac acataatttg aatcaacaca accacccaca gcctaattat 10860 tagcatcatc cctctactat tttttaacca aatcaacaac aacctattta gctgttcccc 10920 aaccttttcc tccgaccccc taacaacccc cctcctaata ctaactacct gactcctacc 10980 cctcacaatc atggcaagcc aacgccactt atccagtgaa ccactatcac gaaaaaaact 11040 ctacctctct atactaatct ccctacaaat ctccttaatt ataacattca cagccacaga 11100 actaatcata ttttatatct tcttcgaaac cacacttatc cccaccttgg ctatcatcac 11160 ccgatgaggc aaccagccag aacgcctgaa cgcaggcaca tacttcctat tctacaccct 11220 agtaggctcc cttcccctac tcatcgcact aatttacact cacaacaccc taggctcact 11280 aaacattcta ctactcactc tcactgccca agaactatca aactcctgag ccaataactt 11340 aatatgacta gcttacacaa tagcttttat agtaaagata cctctttacg gactccactt 11400 atgactccct aaagcccatg tcgaagcccc catcgctggg tcaatagtac ttgccgcagt 11460 actcttaaaa ctaggcggct atggtataat acgcctcaca ctcattctca accccctgac 11520 aaaacacata gcctacccct tccttgtact atccctatga ggcataatta taacaagctc 11580 catctgccta cgacaaacag acctaaaatc gctcattgca tactcttcaa tcagccacat 11640 agccctcgta gtaacagcca ttctcatcca aaccccctga agcttcaccg gcgcagtcat 11700 tctcataatc gcccacgggc ttacatcctc attactattc tgcctagcaa actcaaacta 11760 cgaacgcact cacagtcgca tcataatcct ctctcaagga cttcaaactc tactcccact 11820 aatagctttt tgatgacttc tagcaagcct cgctaacctc gccttacccc ccactattaa 11880 cctactggga gaactctctg tgctagtaac cacgttctcc tgatcaaata tcactctcct 11940 acttacagga ctcaacatac tagtcacagc cctatactcc ctctacatat ttaccacaac 12000 acaatggggc tcactcaccc accacattaa caacataaaa ccctcattca cacgagaaaa 12060 caccctcatg ttcatacacc tatcccccat tctcctccta tccctcaacc ccgacatcat 12120 taccgggttt tcctcttgta aatatagttt aaccaaaaca tcagattgtg aatctgacaa 12180 cagaggctta cgacccctta tttaccgaga aagctcacaa gaactgctaa ctcatgcccc 12240 catgtctaac aacatggctt tctcaacttt taaaggataa cagctatcca ttggtcttag 12300 gccccaaaaa ttttggtgca actccaaata aaagtaataa ccatgcacac tactataacc 12360 accctaaccc tgacttccct aattcccccc atccttacca ccctcgttaa ccctaacaaa 12420 aaaaactcat acccccatta tgtaaaatcc attgtcgcat ccacctttat tatcagtctc 12480 ttccccacaa caatattcat gtgcctagac caagaagtta ttatctcgaa ctgacactga 12540 gccacaaccc aaacaaccca gctctcccta agcttcaaac tagactactt ctccataata 12600 ttcatccctg tagcattgtt cgttacatgg tccatcatag aattctcact gtgatatata 12660 aactcagacc caaacattaa tcagttcttc aaatatctac tcatcttcct aattaccata 12720 ctaatcttag ttaccgctaa caacctattc caactgttca tcggctgaga gggcgtagga 12780 attatatcct tcttgctcat cagttgatga tacgcccgag cagatgccaa cacagcagcc 12840 attcaagcaa tcctatacaa ccgtatcggc gatatcggtt tcatcctcgc cttagcatga 12900 tttatcctac actccaactc atgagaccca caacaaatag cccttctaaa cgctaatcca 12960 agcctcaccc cactactagg cctcctccta gcagcagcag gcaaatcagc ccaattaggt 13020 ctccacccct gactcccctc agccatagaa ggccccaccc cagtctcagc cctactccac 13080 tcaagcacta tagttgtagc aggaatcttc ttactcatcc gcttccaccc cctagcagaa 13140 aatagcccac taatccaaac tctaacacta tgcttaggcg ctatcaccac tctgttcgca 13200 gcagtctgcg cccttacaca aaatgacatc aaaaaaatcg tagccttctc cacttcaagt 13260 caactaggac tcataatagt tacaatcggc atcaaccaac cacacctagc attcctgcac 13320 atctgtaccc acgccttctt caaagccata ctatttatgt gctccgggtc catcatccac 13380 aaccttaaca atgaacaaga tattcgaaaa ataggaggac tactcaaaac catacctctc 13440 acttcaacct ccctcaccat tggcagccta gcattagcag gaataccttt cctcacaggt 13500 ttctactcca aagaccacat catcgaaacc gcaaacatat catacacaaa cgcctgagcc 13560 ctatctatta ctctcatcgc tacctccctg acaagcgcct atagcactcg aataattctt 13620 ctcaccctaa caggtcaacc tcgcttcccc acccttacta acattaacga aaataacccc 13680 accctactaa accccattaa acgcctggca gccggaagcc tattcgcagg atttctcatt 13740 actaacaaca tttcccccgc atcccccttc caaacaacaa tccccctcta cctaaaactc 13800 acagccctcg ctgtcacttt cctaggactt ctaacagccc tagacctcaa ctacctaacc 13860 aacaaactta aaataaaatc cccactatgc acattttatt tctccaacat actcggattc 13920 taccctagca tcacacaccg cacaatcccc tatctaggcc ttcttacgag ccaaaacctg 13980 cccctactcc tcctagacct aacctgacta gaaaagctat tacctaaaac aatttcacag 14040 caccaaatct ccacctccat catcacctca acccaaaaag gcataattaa actttacttc 14100 ctctctttct tcttcccact catcctaacc ctactcctaa tcacataacc tattcccccg 14160 agcaatctca attacaatat atacaccaac aaacaatgtt caaccagtaa ctactactaa 14220 tcaacgccca taatcataca aagcccccgc accaatagga tcctcccgaa tcaaccctga 14280 cccctctcct tcataaatta ttcagcttcc tacactatta aagtttacca caaccaccac 14340 cccatcatac tctttcaccc acagcaccaa tcctacctcc atcgctaacc ccactaaaac 14400 actcaccaag acctcaaccc ctgaccccca tgcctcagga tactcctcaa tagccatcgc 14460 tgtagtatat ccaaagacaa ccatcattcc ccctaaataa attaaaaaaa ctattaaacc 14520 catataacct cccccaaaat tcagaataat aacacacccg accacaccgc taacaatcaa 14580 tactaaaccc ccataaatag gagaaggctt agaagaaaac cccacaaacc ccattactaa 14640 acccacactc aacagaaaca aagcatacat cattattctc gcacggacta caaccacgac 14700 caatgatatg aaaaaccatc gttgtatttc aactacaaga acaccaatga ccccaatacg 14760 caaaattaac cccctaataa aattaattaa ccactcattc atcgacctcc ccaccccatc 14820 caacatctcc gcatgatgaa acttcggctc actccttggc gcctgcctga tcctccaaat 14880 caccacagga ctattcctag ccatgcacta ctcaccagac gcctcaaccg ccttttcatc 14940 aatcgcccac atcactcgag acgtaaatta tggctgaatc atccgctacc ttcacgccaa 15000 tggcgcctca atattcttta tctgcctctt cctacacatc gggcgaggcc tatattacgg 15060 atcatttctc tactcagaaa cctgaaacat cggcattatc ctcctgcttg caactatagc 15120 aacagccttc ataggctatg tcctcccgtg aggccaaata tcattctgag gggccacagt 15180 aattacaaac ttactatccg ccatcccata cattgggaca gacctagttc aatgaatctg 15240 aggaggctac tcagtagaca gtcccaccct cacacgattc tttacctttc acttcatctt 15300 gcccttcatt attgcagccc tagcaacact ccacctccta ttcttgcacg aaacgggatc 15360 aaacaacccc ctaggaatca cctcccattc cgataaaatc accttccacc cttactacac 15420 aatcaaagac gccctcggct tacttctctt ccttctctcc ttaatgacat taacactatt 15480 ctcaccagac ctcctaggcg acccagacaa ttatacccta gccaacccct taaacacccc 15540 tccccacatc aagcccgaat gatatttcct attcgcctac acaattctcc gatccgtccc 15600 taacaaacta ggaggcgtcc ttgccctatt actatccatc ctcatcctag caataatccc 15660 catcctccat atatccaaac aacaaagcat aatatttcgc ccactaagcc aatcacttta 15720 ttgactccta gccgcagacc tcctcattct aacctgaatc ggaggacaac cagtaagcta 15780 cccttttacc atcattggac aagtagcatc cgtactatac ttcacaacaa tcctaatcct 15840 aataccaact atctccctaa ttgaaaacaa aatactcaaa tgggcctgtc cttgtagtat 15900 aaactaatac accagtcttg taaaccggag atgaaaacct ttttccaagg acaaatcaga 15960 gaaaaagtct ttaactccac cattagcacc caaagctaag attctaattt aaactattct 16020 ctgttctttc atggggaagc agatttgggt accacccaag tattgactca cccatcaaca 16080 accgctatgt atttcgtaca ttactgccag ccaccatgaa tattgtacgg taccataaat 16140 acttgaccac ctgtagtaca taaaaaccca atccacatca aaaccccctc cccatgctta 16200 caagcaagta cagcaatcaa ccctcaacta tcacacatca actgcaactc caaagccacc 16260 cctcacccac taggatacca acaaacctac ccacccttaa cagtacatag tacataaagc 16320 catttaccgt acatagcaca ttacagtcaa atcccttctc gtccccatgg atgacccccc 16380 tcagataggg gtcccttgac caccatcctc cgtgaaatca atatcccgca caagagtgct 16440 actctcctcg ctccgggccc ataacacttg ggggtagcta aagtgaactg tatccgacat 16500 ctggttccta cttcagggtc ataaagccta aatagcccac acgttcccct taaataagac 16560 atcacgatg 16569 11 17 DNA B19 Virus 11 tggtctggga tgaaggt 17 12 23 DNA B19 virus 12 ccattttagg cgggcaaccc acc 23 13 21 DNA B19 virus 13 tggaagtgta gctgtgcctg g 21 14 23 DNA B19 virus 14 agaatcattt gtcggaagct cag 23 15 20 DNA B19 virus 15 acaagcctgg gcaagttagc 20 16 21 DNA B19 virus 16 acaatgccag tggaaaggag g 21 17 23 DNA B19 virus 17 ctttaacaca tgaagaccat gca 23 18 28 DNA B19 virus 18 cctctcaaaa cactagaata tccttacg 28 19 27 DNA B19 virus 19 cagccatacc accactggga cacagat 27 20 23 DNA B19 virus 20 aatgccattt ctcatggtca gac 23 21 20 DNA B19 virus 21 gcaaaacaac accacaggca 20 22 22 DNA Hepatitis B virus 22 caacctccaa tcactcacca ac 22 23 24 DNA Hepatitis B virus 23 cctccaattt gtcctggtta tcgc 24 24 23 DNA Hepatitis B virus 24 gtgtctgcgg cgttttatca tat 23 25 23 DNA Hepatitis B virus 25 tgtttggctt tcagctatat gga 23 26 22 DNA Hepatitis B virus 26 aattgtgggt cttttgggct tt 22 27 18 DNA Hepatitis B virus 27 ttctccgtct gccgttcc 18 28 21 DNA Hepatitis B virus 28 accagcacca tgcaactttt t 21 29 25 DNA Hepatitis B virus 29 tttttcacct ctgcctaatc atctc 25 30 16 DNA Hepatitis B virus 30 tcccactgtt caagcc 16 31 21 DNA Hepatitis B virus 31 acctcaccat accgcactca g 21 32 25 DNA Hepatitis B virus 32 gggaattgat gactctagct acctg 25 33 22 DNA Hepatitis B virus 33 atcctgaatg gcaaactcct tc 22 34 20 DNA Hepatitis B virus 34 gagaaaccac acgtagcgca 20 35 16 DNA Hepatitis B virus 35 cacccctcca cacggc 16 36 27 DNA Hepatitis B virus 36 atctctccac ctctaagaga cagtcat 27 37 22 DNA Porcine parvovirus 37 cctcacaaaa cggcaagtac tg 22 38 36 DNA Porcine parvovirus 38 acctaagtcc aagtgactgc tactggttca tacagc 36 39 29 DNA Porcine parvovirus 39 gttaataatg caatgcaaag tacctctaa 29 40 25 DNA Porcine parvovirus 40 aaagacaact gaaagagagc atgga 25 41 30 DNA Porcine parvovirus 41 tctcagctca gattctggct tcatgacaaa 30 42 22 DNA Porcine parvovirus 42 caattctatt tcatgggcca gc 22 43 17 DNA Porcine parvovirus 43 cgtggagcga gccaaca 17 44 28 DNA Porcine parvovirus 44 ctgcacttaa ctccaacacc gccagatt 28 45 24 DNA Porcine parvovirus 45 gcaatacgga caccaagtcc aact 24 46 23 DNA Porcine parvovirus 46 gaggtaagaa gatcgccgag aaa 23 47 28 DNA Porcine parvovirus 47 aacctcacca ccaaccaaaa tatataat 28 48 29 DNA Porcine parvovirus 48 actactaact gaacctacca cagaaggag 29 49 26 DNA Porcine parvovirus 49 cttttacctt cagatccaat aggagg 26 50 18 DNA Sindbis virus 50 gcgtgcggac cctgtact 18 51 25 DNA Sindbis virus 51 attggcttcg acaccaccca gttca 25 52 20 DNA Sindbis virus 52 ttctcggcta tggcaggttc 20 53 25 DNA Sindbis virus 53 gtttatttct ccgtaggatc gacac 25 54 20 DNA Sindbis virus 54 aaaactgctg caggtctcgg 20 55 22 DNA Sindbis virus 55 gaaatcgata ttacaggggc ca 22 56 21 DNA Sindbis virus 56 gcattaagtt tttcggcatg g 21 57 18 DNA Sindbis virus 57 cgattggcat agccggtg 18 58 21 DNA West Nile virus 58 tcagcgatct ctccaccaaa g 21 59 22 DNA West Nile virus 59 tgcccgacca tgggggaagc cc 22 60 21 DNA West Nile virus 60 caatgacaaa cgtgctgacc c 21 61 20 DNA West Nile virus 61 gctagtcctg gtgtttgggg 20 62 24 DNA Escherichia coli 16S Ribosomal RNA 62 agagtttgat catggctcag attg 24 63 24 DNA Escherichia coli 16S Ribosomal RNA 63 ctggcggcag gcctaacaca tgca 24 64 21 DNA Escherichia coli 16S Ribosomal RNA 64 aataccgcat aacgtcgcaa g 21 65 20 DNA Escherichia coli 16S Ribosomal RNA 65 gatgcaacgc gaagaacctt 20 66 23 DNA Escherichia coli 16S Ribosomal RNA 66 gactggggtg aagtcgtaac aag 23 67 24 DNA Escherichia coli 16S Ribosomal RNA 67 gtaacaaggt aaccgtaggg gaac 24 68 19 DNA Escherichia coli 16S Ribosomal RNA 68 gcggttggat cacctcctt 19 69 22 DNA Escherichia coli 23S Ribosomal RNA 69 ccgatagtga accagtaccg tg 22 70 25 DNA Escherichia coli 23S Ribosomal RNA 70 atgttgaaaa attagcggat gactt 25 71 18 DNA Escherichia coli 23S Ribosomal RNA 71 gcactgtttc ggcaaggg 18 72 18 DNA Escherichia coli 23S Ribosomal RNA 72 gccggaagac caagggtt 18 73 24 DNA Escherichia coli 23S Ribosomal RNA 73 ggccgtaact ataacggtcc taag 24 74 26 DNA Escherichia coli 23S Ribosomal RNA 74 gataagtgct gaaagcatct aagcac 26 75 25 DNA Yeast (S. cerevisiae) 75 ctgccagtag tcatatgctt gtctc 25 76 36 DNA Yeast (S. cerevisiae) 76 tacagtgaaa ctgcgaatgg ctcattaaat cagtta 36 77 27 DNA Yeast (S. cerevisiae) 77 taatacatgc ttaaaatctc gaccctt 27 78 24 DNA Yeast (S. cerevisiae) 78 gtcttcggac tctttgatga ttca 24 79 17 DNA Yeast (S. cerevisiae) 79 gcagccgcgg taattcc 17 80 24 DNA Yeast (S. cerevisiae) 80 gctgaaactt aaaggaattg acgg 24 81 23 DNA Yeast (S. cerevisiae) 81 tggaagtttg aggcaataac agg 23 82 19 DNA Yeast (S. cerevisiae) 82 tgaacctgcg gaaggatca 19 83 24 DNA Yeast 25S Ribosomal RNA 83 aagcatatca ataagcggag gaaa 24 84 20 DNA Yeast 25S Ribosomal RNA 84 ctctggtgga ggctcgtagc 20 85 18 DNA Yeast 25S Ribosomal RNA 85 aatggatggc gctcaagc 18 86 25 DNA Yeast 25S Ribosomal RNA 86 tgaaaatcca caggaaggaa tagtt 25 87 21 DNA Yeast 25S Ribosomal RNA 87 ctaagggtcg ggtagtgagg g 21 88 20 DNA Yeast 25S Ribosomal RNA 88 agaaattcaa ccaagcgcga 20 89 19 DNA Yeast 25S Ribosomal RNA 89 atgtcatttt gcgtgggga 19 90 23 DNA Human mitochondrial DNA 90 gttcaccctc taaatcacca cga 23 91 23 DNA Human mitochondrial DNA 91 caagcacgca gcaatgcagc tca 23 92 26 DNA Human mitochondrial DNA 92 ggaaacagca gtgattaacc tttagc 26 93 28 DNA Human mitochondrial DNA 93 gactacgaaa gtggctttaa catatctg 28 94 24 DNA Human mitochondrial DNA 94 tagagtgctt agttgaacag ggcc 24 95 24 DNA Human mitochondrial DNA 95 taggcgatag aaattgaaac ctgg 24 96 21 DNA Human mitochondrial DNA 96 tttgttaaga tggcagagcc c 21 97 20 DNA Human mitochondrial DNA 97 agaatcgaac ccatccctga 20 98 19 DNA Human mitochondrial DNA 98 tttcaccgta ggtggcctg 19 99 20 DNA Human mitochondrial DNA 99 aatcgctgtc gccttaatcc 20 

What is claimed is:
 1. A method for determining the level of potentially active biological pathogens in a biological material, said method comprising: (i) adding to said biological material an effective amount of at least two nucleic acid primer pairs, wherein a first nucleic acid primer pair hybridizes under stringent conditions to a first target nucleic acid sequence found in said biological pathogen and a second nucleic acid primer pair hybridizes under stringent conditions to a second target nucleic acid sequence found in said biological pathogen, and further wherein said first target nucleic acid sequence and said second target nucleic acid sequence are not identical and said second target nucleic acid sequence contains more nucleic acid residues than said first target nucleic acid sequence; (ii) amplifying said target nucleic acid sequences by polymerase chain reaction, said polymerase chain reaction comprising adding at least one polymerase to said biological material containing said nucleic acid primer pairs to form an amplification mixture and thermally cycling said amplification mixture between at least one denaturation temperature and at least one elongation temperature for a period of time sufficient to amplify said target nucleic acid sequences; and (iii) detecting and quantifying said first and second target nucleic acid sequences, wherein the quantity of said first target nucleic acid sequence is proportional to the number of said biological pathogens in said biological material and the quantity of said second target nucleic acid sequence is proportional to the number of potentially active biological pathogens in said biological material.
 2. The method according to claim 1, wherein said first target nucleic acid sequence contains between about 50 and about 500 nucleic acid residues
 3. The method according to claim 1, wherein said second target nucleic acid sequence contains between about 500 and about 50,000 nucleic acid residues.
 4. The method according to claim 1, wherein the nucleic acid sequence of said first target nucleic acid sequence and the nucleic acid sequence of said second target nucleic acid sequence contain at least 16 contiguous nucleic acid residues in common.
 5. The method according to claim 1, wherein said step (i) further comprises adding at least one nucleic acid probe to said biological material.
 6. The method according to claim 5, wherein said nucleic acid probe is selected from the group consisting of 5′ nuclease probes, hairpin probes, adjacent probes, sunrise probes and scorpion probes.
 7. The method according to claim 1, wherein, prior to step (i), said biological material has been subjected to a process that alters at least one wild-type nucleic acid sequence in said biological pathogen.
 8. The method according to claim 7, wherein said process comprises irradiating said biological material with gamma radiation.
 9. The method according to claim 1, wherein one of said first pair of nucleic acid primers and one of said second pair of nucleic acid primers are substantially identical.
 10. The method according to claim 1, wherein neither of said first pair of nucleic acid primers is substantially identical to either of said second pair of nucleic acid primers.
 11. The method according to claim 1, wherein said first pair of nucleic acid primers and said second pair of nucleic acid primers are substantially identical.
 12. The method according to claim 5, wherein said nucleic acid probe contains at least 16 nucleic acid residues.
 13. The method according to claim 1, wherein said biological pathogen is selected from the group consisting of bacteria, viruses, fungi and single cell parasites.
 14. The method according to claim 1, wherein said first target nucleic acid sequence is at least 30% homologous to a wild-type nucleic acid sequence found in said biological pathogen.
 15. The method according to claim 1, wherein said second target nucleic acid sequence is at least 30% homologous to a wild-type nucleic acid sequence found in said biological pathogen.
 16. The method according to claim 14 or 15, wherein said biological pathogen is selected from the group consisting of Aspergillus, Candida, Histoplasma, Saccharomyces, Coccidioides and Cryptococcus.
 17. The method according to claim 14 or 15, wherein said biological pathogen is selected from the group consisting of Escherichia, Bacillus, Campylobacter, Helicobacter, Lysteria, Clostridium, Streptococcus, Enterococcus, Staphylococcus, Brucella, Haemophilus, Salmonella, Yersinia, Pseudomonas, Serratia, Enterobacter, Kebsiella, Proteus, Citrobacter, Corynebacterium, Propionibacterium and Coxiella.
 18. The method according to claim 14 or 15, wherein said biological pathogen is selected from the group consisting of Adeno-associated Virus (AAV), California Encephalitis Virus, Coronavirus, Coxsackievirus-A, Coxsackievirus-B, Eastern Equine Encephalitis Virus (EEEV), Echovirus, Hantavitus, Hepatitis A Virus (HAV), Hepatitis C Virus (HCV), Hepatitis Delta Virus (HDV), Hepatitis E Virus (HEV), Hepatitis G Virus (HGV), Human Immunodeficiency Virus (HIV), Human T-lymphotrophic Virus (HTLV), Influenza Virus (Flu Virus), Measles Virus (Rubeola), Mumps Virus, Norwalk Virus, Parainfluenza Virus, Polio virus, Rabies Virus, Respiratory Syncytial Virus, Rhinovirus, Rubella Virus, Saint Louis Encephalitis Virus, Western Equine Encephalitis Virus (WEEV), Yellow Fever Virus, Adenovirus, Cytomegalovirus (CMV), Epstein-Barr Virus (EBV), Hepatitis B Virus (HBV), Herpes Simplex Virus 1 (HHV1), Herpes Simplex Virus 2 (HHV2), Molluscum contagiosum, Papilloma Virus (HPV), Smallpox Virus (Variola), Vaccinia Virus, Venezuelan Equine Encephalitis Virus (VEEV), Ebola Virus, West Nile Virus, Human Parvovirus B19 and Rotavirus.
 19. The method according to claim 15, wherein said wild-type nucleic acid sequence comprises the 16S ribosomal RNA gene coding sequence.
 20. The method according to claim 15, wherein said wild-type nucleic acid sequence comprises the 16S ribosomal RNA gene coding sequence and a portion of the 23S ribosomal RNA gene coding sequence.
 21. The method according to claim 15, wherein said wild-type nucleic acid sequence comprises the 16S ribosomal RNA gene coding sequence and a portion of the 23S ribosomal RNA gene coding sequence and the non-coding sequence therebetween.
 22. The method according to claim 14, wherein said wild-type nucleic acid sequence comprises the 18S ribosomal RNA gene coding sequence.
 23. The method according to claim 14, wherein said wild-type nucleic acid sequence comprises the 18S ribosomal RNA gene coding sequence and the 5.8S ribosomal RNA gene coding sequence.
 24. The method according to claim 14, wherein said wild-type nucleic acid sequence comprises the 18S ribosomal RNA gene coding sequence and the 5.8S ribosomal RNA gene coding sequence and the non-coding sequence therebetween.
 25. The method according to claim 14, wherein said wild-type nucleic acid sequence comprises the 18S ribosomal RNA gene coding sequence and the 5.8S ribosomal RNA gene coding sequence and a portion of the 28S ribosomal RNA gene coding sequence.
 26. The method according to claim 14, wherein said wild-type nucleic acid sequence comprises the 18S ribosomal RNA gene coding sequence and the 5.8S ribosomal RNA gene coding sequence and a portion of the 28S ribosomal RNA gene coding sequence and the non-coding sequences therebetween.
 27. The method according to claim 1, wherein said polymerase is a thermostable polymerase.
 28. The method according to claim 27, wherein said thermostable polymerase is a Taq polymetase.
 29. The method according to claim 1, wherein said polymerase chain reaction thermally cycling said amplification mixture between at least one denaturation temperature, at least one annealing temperature and at least one elongation temperature for a period of time sufficient to amplify said target nucleic acid sequence.
 30. The method according to claim 7, wherein said process fragments said at least one wild-type nucleic acid sequence in said biological pathogen.
 31. The method according to claim 7, wherein said process cross-links said at least one wild-type nucleic acid sequence in said biological pathogen.
 32. The method according to claim 7, wherein said process covalently modifies said at least one wild-type nucleic acid sequence in said biological pathogen.
 33. The method according to claim 1, wherein said biological material is selected from the group consisting of: cells; tissues; blood or blood components; proteins; enzymes; immunoglobulins; botanicals; and food.
 34. The method according to claim 1, wherein said biological material is selected from the group consisting of ligaments; tendons; nerves; bone; teeth; skin grafts; bone marrow; heart valves; cartilage; corneas; arteries and veins; organs; lipids; carbohydrates; collagen; chitin and its derivatives; forensic samples, mummified material; human or animal remains; stem cells; islet of Langerhans cells; cells for transplantation; red blood cells; white blood cells; and platelets. 